Antibodies against monocyte chemotactic proteins

ABSTRACT

The invention provides antibodies that bind to a plurality of β-chemokines, particularly monocyte chemotactic proteins MCP-1, MCP-2 and MCP-3. The invention also provides cells producing the antibodies, and methods of making and using the same.

RELATED APPLICATIONS

This is a division of application Ser. No. 13/156,959, filed Jun. 9,2011, which is a continuation of application Ser. No. 12/612,087, filedNov. 4, 2009, which issued as U.S. Pat. No. 7,972,597 on Jul. 5, 2011,which is a division of U.S. patent application Ser. No. 12/171,791,filed Jul. 11, 2008, issued as U.S. Pat. No. 7,632,501 on Dec. 15, 2009,which is a division of U.S. patent application Ser. No. 10/855,013,filed May 27, 2004, issued as U.S. Pat. No. 7,405,277 on Jul. 29, 2008,which is a continuation of International Patent ApplicationPCT/US02/038229, filed Nov. 27, 2002, which claims priority to U.S.Provisional Application Nos. 60/343,391, filed Nov. 30, 2001; 60/383277,filed May 24, 2002, and 60/400,469, filed Aug. 1, 2002, all of which areincorporated herein by reference.

BACKGROUND OF THE INVENTION

The invention relates to antibodies that specifically recognizeβ-chemokines. Specifically, the invention is drawn to antibodies thatspecifically recognize monocyte chemotactic proteins designated MCP-1,MCP-2 and MCP-3, particularly antibodies that specifically bind to MCP-1and MCP-2; MCP-2 and MCP-3, MCP-1 and MCP-3; and MCP-1, MCP-2 and MCP-3.

“Chemokines,” which take their name from chemotactic cytokines, aresmall secreted polypeptides that regulate movement of immune cells intotissues (Baggiolini et al. (1994) Adv. Immunol. 55:97-179; Oppenheim etal. (1991) Ann Rev. Immunol. 9:617-648). Chemokines are assigned tothree different families based on the number and position of conservedcysteine residues (Van Coillie et al. (1999) Cytokine & Growth FactorRev. 10:61-86). The α and β chemokines each contain four conservedcysteine residues. The first two cysteines of the α chemokines areseparated by a single amino acid, thus containing a CXC amino acidmotif. The first two conserved cysteines of the β chemokines areadjacent. Thus, the β chemokines are also known as C—C chemokines. Bycontrast, lymphotactin is the sole member of the third family ofchemokines, and contains only the second and fourth conserved cysteineresidues. Interestingly, in humans, α chemokines are all encoded bygenes on chromosome 4, β chemokines are all encoded by genes onchromosome 17, and lymphotaxin is encoded by genes on chromosome 1.

The β-chemokines form a gradient that serves as a chemoattractant andpotential proliferation signal for immune and other cells such asmonocytes, macrophages, basophils, eosinophils, T lymphocytes andfibroblasts. MCP-1, MCP-2 and MCP-3 share sequence homology with oneanother at the amino acid level. Through interaction with specificreceptors, termed C—C chemokine receptors (CCR) which are G-proteincoupled, seven transmembrane receptors (Rossi and Zlotnik (2000) Ann.Rev. Immunol. 18:217-242), the β-chemokines regulate the expression ofadhesion molecules on endothelial cells and thereby indirectly affectdiapedesis and extravasation of immune cells from the circulation intotissues. There are ten different CCRs (CCR1 through CCR10). CCR2 acts asa receptor for MCP-1, MCP-2, MCP-3, and MCP-4 (Rossi and Zlotnik (2000)Ann. Rev. Immunol. 18:217-242). However, all human MCPs have been shownto interact with more than one receptor (Van Coillie et al. (1999)Cytokine & Growth Factor Rev. 10:61-86).

Human MCP-1, MCP-2 and MCP-3 all have chemotactic activity for a varietyof cell types, including T lymphocytes and monocytes (Van Coillie et al.(1999) Cytokine & Growth Factor Rev. 10:61-86). Other shared functionsof MCP-1, MCP-2, and MCP-3 include induction of N-acetylβ-D-glucosaminidase release, gelatinase B release, and granzyme Arelease which are believed to help the cells digest the extracellularmatrix components necessary to enable them to migrate into tissues (VanCoillie et al. (1999) Cytokine & Growth Factor Rev. 10:61-86). Inaddition, MCP-1 and MCP-3 share various functions, such as induction ofarachidonic acid release and stimulation of a respiratory burst (VanCoillie et al. (1999) Cytokine & Growth Factor Rev. 10:61-86).

MCP-1-specific antibodies have previously been described in theliterature (WO 01/89582, WO 01/89565, Luo et al. (1994) J Immunol153:3708-16; Traynor, et al. (2002) J Immunol 168:4659-66). CertainMCP-1 antibodies have been described as binding MCP-1 and MCP-3,specifically the MRHAS domain of MCP-1 and MCP-3 (WO 95/09232). Inaddition, a human anti-MCP-1 antibody has also been described (WO02/02640). There is a need in the art to identify antibodies which canbe used to manipulate β-chemokines in general, and to specificallymodulate the activity of multiple chemokines, e.g., MCP-1 and MCP-2 orMCP-3.

SUMMARY OF THE INVENTION

The invention provides antibodies that specifically bind to a variety ofβ-chemokines, and monocyte chemotactic proteins in particular. Theinvention encompasses antibodies against individual β-chemokines andagainst β-chemokines in general. The so-called “pan-antibodies” of theinvention bind to more than one of the β-chemokines, i.e., monocytechemotactic proteins, MCP-1, MCP-2, and MCP-3.

The invention further provides antibodies that specifically bind to bothMCP-1 and MCP-2, both MCP-2 and MCP-3, both MCP-1 and MCP-3 (to a regionother than an MCP MRHAS motif), or to all three of MCP-1, MCP-2 andMCP-3.

In some embodiments, the antibodies are monoclonal antibodies. Themonoclonal antibodies of the invention are preferably mammalianantibodies. In some embodiments, the antibodies are human antibodies.

In some embodiments, the antibodies are selected from the groupconsisting of 11K2.1 (ATCC Accession No. PTA-3987), 6D21.1 (ATCCAccession No. PTA-3989), 4N4.1 (ATCC Accession No. PTA-3994), 5A13.1(ATCC Accession No. PTA-3995), 7H1.1 (ATCC Accession No. PTA-3985),1A1.1 (ATCC Accession No. PTA-3990) 6I5.1 (ATCC Accession No. PTA-3986),2O24.1 (ATCC Accession No. PTA-3993), 9B11.1 (ATCC Accession No.PTA-3992), 9B12.1 (ATCC Accession No. PTA-3996), 9C11.1 (ATCC AccessionNo. PTA-3988), and 12F15.1 (ATCC Accession No. PTA-3991).

The invention further provides antibody fragments that specifically bindto a variety of β-chemokines, particularly monocyte chemotacticproteins, including both MCP-1 and MCP-2, both MCP-2 and MCP-3, bothMCP-1 and MCP-3 (to a region other than an MCP MRHAS motif), or to allthree of MCP-1, MCP-2 and MCP-3. The antibody fragments include Fab,Fab′, F(ab′)₂, and F_(v). The invention also embraces single chainantibodies, chimeric antibodies and humanized antibodies.

In some embodiments, the antigen-binding fragments of the antibodies areantigen-binding fragments of antibodies selected from the groupconsisting of 11K2.1 (ATCC Accession No. PTA-3987), 6D21.1 (ATCCAccession No. PTA-3989), 4N4.1 (ATCC Accession No. PTA-3994), 5A13.1(ATCC Accession No. PTA-3995), 7H1.1 (ATCC Accession No. PTA-3985),1A1.1 (ATCC Accession No. PTA-3990), 6I5.1 (ATCC Accession No.PTA-3986), 2O24.1 (ATCC Accession No. PTA-3993), 9B11.1 (ATCC AccessionNo. PTA-3992), 9B12.1 (ATCC Accession No. PTA-3996), 9C11.1 (ATCCAccession No. PTA-3988), and 12F15.1 (ATCC Accession No. PTA-3991).

The invention further provides hybridoma and transformed cells producingany of the antibodies or antigen-binding fragments described herein.

In some embodiments, the antibodies and antigen-binding fragmentsthereof may be chemically modified to provide a desired effect. In someembodiments the antibodies or antigen-binding fragments thereof areconjugated to polyethylene glycol or albumen.

In some embodiments, the antibodies and antigen-binding fragmentsthereof may be conjugated to a toxins, or radioisotopes.

The invention further provides in vitro immunoassays for detectingβ-chemokines in samples.

The invention also provides therapeutic compositions containing at leastone of the anti-β-chemokine antibodies provided herein in apharmaceutically acceptable carrier.

The invention also provides method of producing the anti-β-chemokineantibodies and anti-β-chemokine antibody producing cells providedherein.

The invention also provides methods of inhibiting the activity ofβ-chemokines comprising administering to an animal compositioncomprising a pharmaceutically acceptable carrier and an antibody thatbinds to at least one β-chemokine or antigen-binding fragment thereof.In some embodiment, the antibody binds MCP-1 and MCP-2. In otherembodiments, the antibody binds MCP-2 and MCP-3. In other embodiments,the antibody binds MCP-1 and MCP-3 to a region other than an MRHASmotif. In other embodiments, the antibody binds MCP-1, MCP-2 and MCP-3.

The invention also provides methods of blocking chemotaxis comprisingadministering to an animal a composition comprising a pharmaceuticallyacceptable carrier and a antibody or antigen-binding fragment that bindsto at least one β-chemokine. In some embodiments, the antibody bindsMCP-1 and MCP-2. In other embodiments, the antibody binds MCP-2 andMCP-3. In other embodiment, the antibody binds MCP-1 and MCP-3 to aregion other than an MRHAS motif. In other embodiments, the antibodybinds MCP-1, MCP-2 and MCP-3.

The invention also provides methods of modulating β-chemokine activityin vitro and in vivo using the antibodies or antigen-binding fragments,and compositions provided herein. As such, the antibodies orantigen-binding fragments of the invention are useful in the treatmentof diseases and disorders including, but not limited to: inflammatoryconditions and pathological conditions associated with MCP.

In certain embodiments, the antibodies or antigen-binding fragmentsthereof of the present invention are useful in the treatment ofglomerulonephritis, scleroderma, cirrhosis, multiple sclerosis, lupusnephritis, atherosclerosis, rheumatoid arthritis, and inflammatory boweldisease.

The invention provides antibodies or antigen-binding fragments thereof,that bind to MCP-1, MCP-2 and/or MCP-3 wherein the antibodies, orantigen-binding fragments thereof, have a Kd for binding to MCP-1, MCP-2and/or MCP-3 of about 1 pM or less, about 0.7 pM or less or about 0.4 pMor less. The invention also provides antibodies or antigen-bindingfragments thereof, that bind to MCP-1, MCP-2 and/or MCP-3 comprising aFab fragment wherein the Fab fragment has a Kd for binding to MCP-1,MCP-2 and/or MCP-3 of about 15 pM or less, about 13 pM or less or about11 pM or less.

The invention provides as antibody, or antigen-binding fragment thereof,comprising a variable heavy chain region as set forth to SEQ ID NO:11and a variable light chain region as set forth in SEQ ID NO:12. In oneembodiment, the antibody is a chimeric antibody. In another embodiment,the antibody is a humanized antibody. In still another embodiment, theantibody of the invention is a Fab fragment.

The invention also provides an isolated polypeptide comprising afragment of SEQ ID NO:11 selected from the grasp consisting of aminoacids 31-35 of SEQ ID NO:11, amino acids 50-66 of SEQ ID NO:11, andamino acids 99-111 of SEQ ID NO:11. In another embodiment, the inventionprovides an isolated polypeptide comprising amino acids 31-35 of SEQ IDNO:11, amino acids 50-66 of SEQ ID NO:11 and amino acids 99-111 of SEQID NO:11. In a further embodiment, the invention provides an isolatedpolypeptide comprising the amino acid sequence of SEQ ID NO:11.

In another embodiment, the invention features an isolated polypeptidecomprising a fragment of SEQ ID NO:12 selected from the group consistingof amino acids 24-39 of SEQ ID NO:12, amino acids 55-61 of in SEQ IDNO:12, and amino acids 94-102 of SEQ ID NO:12. The invention alsofeatures an isolated polypeptide comprising amino acids 24-39 of SEQ IDNO:12, amino acids 55-61 of SEQ ID NO:12, and amino acids 94-102 of SEQID NO:12. In yet another embodiment, the invention provides an isolatedpolypeptide comprising the amino acid sequence of SEQ ID NO:12.

In some embodiments, the invention provides a variant of a polypeptidecomprising the amino acid sequence of SEQ ID NO:11, said variantcomprising at least one conservative amino acid substitution, whereinthe variant retains the ability to bind to MCP-1 with a Kd of about 0.7pM or less. In another embodiment, the invention features a variant of apolypeptide comprising the amino acid sequence of SEQ ID NO:11, saidvariant comprising at least one conservative amino acid substitution,wherein the variant retains the ability to bind MCP-2 with a Kd of about1.2 pM or less. In yet another embodiment, the invention provides avariant of a polypeptide comprising the amino acid sequence of SEQ IDNO:12, said variant comprising at least one conservative amino acidsubstitution, wherein the variant retains the ability to bind MCP-1 witha Kd of about 0.7 pM or less. In still another embodiment, the inventionfeatures a variant of a polypeptide comprising the amino acid sequenceof SEQ ID NO:12, said variant comprising at least one conservative aminoacid substitution, wherein the variant retains the ability to bind MCP-2with a Kd of about 1.2 pM or less.

The invention provides an isolated polypeptide comprising the amino acidsequence of SEQ ID NO:11 and SEQ ID NO:12.

In one embodiment, the invention features an antibody heavy chaincomprising a variable region complementarity determining region (CDR)from the 11K2 antibody heavy chain variable region set forth in SEQ IDNO:27. The invention also features as antibody light chain comprising avariable region complementarity determining region (CDR) from the 11K2antibody light chain variable region set forth in SEQ ID NO:28. Theinvention provides an antibody, or antigen-binding fragment thereof,comprising a variable heavy chain region as set forth in SEQ ID NO:27and a variable light chain region as set forth is SEQ ID NO:28. In oneembodiment of the invention, the antibody is a chimeric antibody. Instill another embodiment, the antibody of the invention is a humanizedantibody. In still another embodiment, the antibody of the invention isa Fab fragment.

The invention provides an isolated polypeptide comprising a fragment ofSEQ ID NO:27 selected from the grasp consisting of amino acids 31-35 ofSEQ ID NO:27, amino acids 50-66 of SEQ ID NO:27 and amino acids 99-106of SEQ ID NO:27.

The invention also features an isolated polypeptide comprising aminoacids 31-35 of SEQ ID NO:27, amino acids 50-66 of SEQ ID NO:27, andamino acids 99-106 of SEQ ID NO:27. Still another feature of theinvention is an isolated polypeptide comprising the amino acid sequenceof SEQ ID NO:27.

In another embodiment, the invention provides an isolated polypeptidecomprising a fragment of SEQ ID NO:28 selected from the group consistingof amino acids 24-34 of SEQ ID NO:28, amino acids 50-56 of in SEQ IDNO:28, and amino acids 89-97 of SEQ ID NO:28. The invention alsoprovides an isolated polypeptide comprising amino acids 24-34 of in SEQID NO:28, amino acids 50-56 of SEQ ID NO:28, and amino acids 89-97 ofSEQ ID NO:28. In still another embodiment, the invention provides anisolated polypeptide comprising the amino acid sequence of SEQ ID NO:28.

In some embodiments, the invention provides a variant of a polypeptidecomprising the amino acid sequence of SEQ ID NO:27, said variantcomprising at least one conservative amino acid substitution, whereinthe variant retains the ability to bind MCP-1 with a Kd of about 0.4 pMor less. The invention also features a variant of a polypeptidecomprising the amino acid sequence of SEQ ID NO:27, said variantcomprising at least one conservative amino acid substitution, whereinthe variant retains the ability to bind MCP-2 with a Kd of about 18 pMor less. In another embodiment, the invention features a variant of apolypeptide comprising the amino acid sequence of SEQ ID NO:28, saidvariant comprising at least one conservative amino acid substitution,wherein the variant retains the ability to bind MCP-1 with a Kd of about0.4 pM or less. In still another embodiment, the invention provides avariant of a polypeptide comprising the amino acid sentence of SEQ IDNO:28, said variant comprising at least one conservative amino acidsubstitution, wherein the variant retains the ability to bind MCP-2 witha Kd of about 18 pM or less.

The invention provides an isolated polypeptide comprising the amino acidsequence of SEQ ID NO:27 and SEQ ID NO:28.

The invention features an isolated nucleic acid encoding theimmunoglobulin of SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:27, and SEQ IDNO:28. The invention also provides isolated nucleic acid moleculesencoding the polypeptide of any one of SEQ ID NO:13, SEQ ID NO:14, SEQID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:29, SEQ IDNO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, and SEQ ID NO:34. Inanother embodiment, the invention provides isolated nucleic acidmolecules comprising SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:25, and SEQ IDNO:26. In one embodiment, the invention features a vector comprising thenucleic acid molecules of the invention. In addition, the inventionfeatures a host cell comprising the nucleic acids of the invention. Theinvention also features a method of producing a antibody or fragmentthereof, comprising culturing the host cell comprising the nucleic acidsof the invention under conditions such that the antibody or fragment isproduced and isolating said antibody from the host cell or culture.

The invention further provides an antibody heavy chain comprisingvariable region complementarity determining regions (CDRs) from the 1A1antibody heavy chain variable region set forth as SEQ ID NO:11. Theinvention further provides an antibody light chain comprising variableregion complementarity determining regions (CDRs) from the 1A1 antibodylight chain satiable region set forth as SEQ ID NO:12.

The invention further provides an antibody heavy chain comprisingvariable region complementarity determining regions (CDRs) from the 11K2antibody heavy chain variable region set forth as SEQ ID NO:27. Theinvention also provides an antibody light chain comprising variableregion complementarity determining regions (CDRs) from the 11K2 antibodylight chain variable region set forth as SEQ ID NO:28.

The invention also features an isolated antibody, or antigen-bindingfragment thereof, which binds a plurality of β-chemokines, wherein saidβ-chemokines comprise MCP-1 and at least one other β-chemokine (e.g.,MCP-2), and wherein said antibody or antigen-binding fragment comprisesat least one of the following CDRs: CDR1, CDR2, or CDR3, from the 1A1heavy chain variable region set forth as SEQ ID NO:11. In anotherembodiment, the invention provides an isolated antibody, orantigen-binding fragment thereof, which binds a plurality ofβ-chemokines, wherein said β-chemokines comprise MCP-1 and at least oneother β-chemokine (e.g., MCP-2), and wherein said antibody orantigen-binding fragment comprises at least one of the following CDRcombinations: CDR1 and CDR2; CDR1 and CDR3; CDR2 and CDR3; and CDR1,CDR2, and CDR3, from the 1A1 heavy chain variable region set forth asSEQ ID NO:11.

The invention provides an isolated antibody, or antigen-binding fragmentthereof, which binds a plurality of β-chemokines, wherein saidβ-chemokines comprise MCP-1 and at least one other β-chemokine (e.g.,MCP-2), and wherein said antibody or antigen-binding fragment comprisesat least one of the following CDRs; CDR1, CDR2, or CDR3, from the 1A1light chain variable region set forth as SEQ ID NO:12. In anotherembodiment, the invention provides an isolated antibody, orantigen-binding fragment thereof, which binds a plurality ofβ-chemokines, wherein said β-chemokines comprise MCP-1 and at least oneother β-chemokine (e.g., MCP-2), and wherein said antibody orantigen-binding fragment comprises at least one of the following CDRcombinations: CDR1 and CDR2; CDR1 and CDR3; CDR2 and CDR3; or CDR1,CDR2, and CDR3, from the 1A1 light chain variable region set forth asSEQ ID NO:12.

The invention features an isolated antibody, or antigen-binding fragmentthereof, which binds a plurality of β-chemokines, wherein saidβ-chemokines comprise MCP-1 and at least one other β-chemokine (e.g.,MCP-2), and wherein said antibody or antigen-binding fragment comprisesat least one of the following CDRs: CDR1, CDR2, or CDR3, from the 11K2heavy chain variable region set forth as SEQ ID NO:27. In an additionalembodiment, the invention features an isolated antibody, orantigen-binding fragment thereof, which binds a plurality ofβ-chemokines, wherein said β-chemokines comprise MCP-1 and at least oneother β-chemokine (e.g., MCP-2), and wherein said antibody orantigen-binding fragment comprises at least one of the following CDRcombinations: CDR1 and CDR2; CDR1 and CDR3; CDR2 and CDR3; or CDR1,CDR2, and CDR3, from the 11K2 heavy chain variable region set forth asSEQ ID NO:27.

The invention also provides an isolated antibody, or antigen-bindingfragment thereof, which binds a plurality of β-chemokines, wherein saidβ-chemokines comprise MCP-1 and at least one other β-chemokine (e.g.,MCP-2), and wherein said antibody or antigen-binding fragment comprisesat least one of the following CDRs: CDR1, CDR2, or CDR3, from the 11K2light chain variable region set forth as SEQ ID NO:28. In anotherembodiment, the invention provides an isolated antibody, orantigen-binding fragment thereof, which binds a plurality ofβ-chemokines, wherein said β-chemokines comprise MCP-1 and at least oneother β-chemokine (e.g., MCP-2), and wherein said antibody orantigen-binding fragment comprises at least one of the following CDRcombinations: CDR1 and CDR2; CDR1 and CDR3; CDR2 and CDR3; or CDR1,CDR2, and CDR3, from the 11K2 light chain variable region set forth asSEQ ID NO:28.

The invention provides an antibody, or antigen-binding fragment thereof,which binds a plurality of β-chemokines, and wherein said β-chemokinescomprise MCP-1 and at least one other β-chemokine (e.g., MCP-2), whereinsaid antibody or antigen-binding fragment thereof comprises a heavychain variable region having a CDR1 domain comprising the sequence setforth as SEQ ID NO:13, a CDR2 domain comprising the sequence set forthas SEQ ID NO:14, and a CDR3 domain comprising the sequence set forth asSEQ ID NO:15. In one embodiment, the antibody of the invention is achimeric antibody.

The invention also provides an antibody or antigen-binding fragmentthereof, which binds a plurality of β-chemokines, and wherein saidβ-chemokines comprise MCP-1 and at least one other β-chemokine (e.g.,MCP-2), wherein said antibody or antigen-binding fragment thereofcomprises a light chain variable region having a CDR1 domain comprisingthe sequence set forth as SEQ ID NO:16, a CDR2 domain comprising thesequence set forth as SEQ ID NO:17, and a CDR3 domain comprising thesequence set forth as SEQ ID NO:18.

The invention further provides an antibody, or antigen-binding fragmentthereof, which binds a plurality of β-chemokines, and wherein saidβ-chemokines comprise MCP-1 and at least one other β-chemokine (e.g.,MCP-2), wherein said antibody or antigen-binding fragment thereofcomprises a heavy chain variable region having a CDR1 domain comprisingthe sequence set forth as SEQ ID NO:13, a CDR2 domain comprising thesequence set forth as SEQ ID NO:14, and a CDR3 domain comprising thesequence set forth as SEQ ID NO:15, and a light chain variable regionhaving a CDR1 domain comprising the sequence set forth as SEQ ID NO:16,a CDR2 domain comprising the sequence set forth as SEQ ID NO:17, and aCDR3 domain comprising the sequence set forth as SEQ ID NO:18.

The invention provides as antibody or antigen-binding fragment thereof,which binds a plurality of β-chemokines, and wherein said β-chemokinescomprise MCP-1 and at least one other β-chemokine (e.g., MCP-2), whereinsaid antibody or antigen-binding fragment thereof comprises a heavychain variable region having a CDR1 domain comprising the sequence setforth as SEQ ID NO:29, a CDR2 domain comprising the sequence set forthas SEQ ID NO:30, and a CDR3 domain comprising the sequence set forth asSEQ ID NO:31.

In addition, the invention provides an antibody, or antigen-bindingfragment thereof, which binds a plurality of β-chemokines, wherein saidβ-chemokines comprise MCP-1 and at least one other β-chemokine (e.g.,MCP-2), and wherein said antibody or antigen-binding fragment thereofcomprises a light chain variable region having a CDR1 domain comprisingthe sequence set forth as SEQ ID NO:32, a CDR2 domain comprising thesequence set forth as SEQ ID NO:33, and a CDR3 domain comprising thesequence set forth as SEQ ID NO:34. In one embodiment, the antibody ofthe invention is a chimeric antibody. In another embodiment, theantibody of the invention is a humanized antibody.

The invention further provides an antibody or antigen-binding fragmentthereof, which binds a plurality of β-chemokines, wherein saidβ-chemokines comprise MCP-1 and at least one other β-chemokine (e.g.,MCP-2), and wherein said antibody or antigen-binding fragment thereofcomprises a heavy chain variable region having a CDR1 domain comprisingthe sequence set forth as SEQ ID NO:29, a CDR2 domain comprising thesequence set forth as SEQ ID NO:30, and a CDR3 domain comprising thesequence set forth as SEQ ID NO:31, and a light chain variable regionhaving a CDR1 domain comprising the sequence set forth as SEQ ID NO:32,a CDR2 domain comprising the sequence set forth as SEQ ID NO:33, and aCDR3 domain comprising the sequence set forth as SEQ ID NO:34. In oneembodiment, the antibody of the invention is a chimeric antibody. Inanother embodiment, the antibody of the invention is a humanizedantibody.

In one embodiment, the invention provides an isolated polypeptideencoding a CDR3 domain comprising an amino acid sequence selected fromthe group consisting of: SEQ ID NO:15, SEQ ID NO:18, SEQ ID NO:31, andSEQ ID NO:34. In another embodiment, the invention features an isolatedpolypeptide encoding a CDR2 domain comprising an amino acid sequenceselected from the group consisting of: SEQ ID NO:14, SEQ ID NO:17, SEQID NO:30, and SEQ ID NO:33. In still another embodiment, the inventionprovides an isolated polypeptide encoding a CDR1 domain comprising anamino acid sequence selected from the group consisting of: SEQ ID NO:13,SEQ ID NO:16, SEQ ID NO:29, and SEQ ID NO:32.

In yet another embodiment, the invention features an antibody heavychain comprising a variable region complementarity determining region(CDR) from the 11K2 antibody heavy chain variable region set forth inSEQ ID NO:27. The invention also features an antibody light chaincomprising a variable region complementarity determining region (CDR)from the 11K2 antibody light chain variable region set forth in SEQ IDNO:28. In still a further embodiment, the invention features an antibodyor antigen-binding fragment thereof, comprising a variable heavy chainregion as set forth in SEQ ID NO:27 and a variable light chain region asset forth in SEQ ID NO:28.

In another embodiment, the invention features an antibody, orantigen-binding fragment thereof, which binds a plurality ofβ-chemokines, wherein said β-chemokines comprise MCP-1 and at least oneother β-chemokine, and wherein said antibody or antigen-binding fragmentcomprises the variable region complementarity determining region (CDR)from the 11K2 heavy chain variable region set forth in SEQ ID NO:27. Theinvention also features an antibody, or antigen-binding fragmentthereof, which binds a plurality of β-chemokines, wherein saidβ-chemokines comprise MCP-1 and at least one other β-chemokine, andwherein said antibody or antigen-binding fragment comprises the variableregion complementarity determining region (CDR) from the 11K2 lightchain variable region set forth in SEQ ID NO:28. In yet anotherembodiment, at least one other β-chemokine is MCP-2.

Another embodiment of the invention includes an antibody, orantigen-binding fragment thereof, which binds a plurality ofβ-chemokines, wherein said β-chemokines comprise MCP-1 and at least oneother β-chemokine, and wherein said antibody or antigen-binding fragmentcomprises a heavy chain variable region having a CDR1 domain comprisingthe sequence set forth in SEQ ID NO:29, a CDR2 domain comprising thesequence set forth in SEQ ID NO:30, and a CDR3 domain comprising thesequence set forth in SEQ ID NO:31. The invention also features anantibody, or antigen-binding fragment thereof, which binds a pluralityof β-chemokines, wherein said β-chemokines comprise MCP-1 and at leastone other β-chemokine, and wherein said antibody or antigen-bindingfragment comprises a light chain variable region having a CDR1 domaincomprising the sequence set forth in SEQ ID NO:32, a CDR2 domaincomprising the sequence set forth in SEQ ID NO:33, and a CDR3 domaincomprising the sequence set forth in SEQ ID NO:34.

In still another embodiment, the indention features an antibody orantigen-binding fragment thereof, which binds a plurality ofβ-chemokines, wherein said β-chemokines comprise MCP-1 and at least oneother β-chemokine, and wherein said antibody or antigen-binding fragmentcomprises a heavy chain variable region having a CDR1 domain comprisingthe sequence set forth in SEQ ID NO:29, a CDR2 domain comprising thesequence set forth in SEQ ID NO:30, and a CDR3 domain comprising thesequence set forth in SEQ ID NO:31, and a light chain variable regionhaving a CDR1 domain comprising the sequence set forth in SEQ ID NO:32,a CDR2 domain comprising the sequence set forth in SEQ ID NO:33, and aCDR3 domain comprising the sequence set forth in SEQ ID NO:34. In oneembodiment, at least one other β-chemokine is MCP-2.

In another embodiment, the antibody or antigen-binding fragment of theinvention is modified to reduce or eliminate potential glycosylationsites. In still another embodiment, the constant region of the antibody,or fragment thereof of the invention is modified to reduce at least oneconstant region-mediated biological effector function relative to anunmodified antibody.

In yet another embodiment, the invention features a method of treating asubject suffering from a disorder selected from the gram consisting ofglomerulonephritis, scleroderma, cirrhosis, multiple sclerosis, lupusnephritis, atherosclerosis, inflammatory bowel disease or rheumatoidarthritis, comprising administering to the subject an antibody orfragment thereof of the invention.

The invention also provides a kit for detecting the presence ofβ-chemokines in a sample comprising reagents for performing animmunoassay; an antibody or antigen-binding fragment thereof thatspecifically binds to a plurality of β-chemokines comprising MCP-1 andat least one other β-chemokine; and reagents for detecting the bindingof said antibody or antigen-binding fragment thereof.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 graphically depicts the results of a chemotaxis assay using 11K2,7F7, 6D21, and 7H1 hybridoma supernatant to inhibit chemotaxis inresponse to MCP-1. Each of the antibodies tested was able to inhibitMCP-1 induced chemotaxis, with 11K2 and 6D21 being the most effective.

FIG. 2 graphically depicts results of a chemotaxis assay using purified11K2, 1A1, D9, and 2O24 to inhibit chemotaxis in response to MCP-1,MCP-2, and a combination of MCP-1/MCP-2. The results show thatchemotaxis to a combination of MCP-1 and MCP-2 is inhibited by 11K2 and1A1.

FIGS. 3A and 3B graphically depict the results of a chemotaxis assay ofcells in response to MCP-1. FIG. 3A graphically depicts results usingmonoclonal antibodies 5D3-F7 (BD Biosciences, Pharmingen, San Diego,CA), 1M11, 3N10, 5J23, and 11K2 in response to 20 ng/mL of MCP-1. FIG.3B graphically depicts results using 20 ng/mL of murine MCP-1(JE) andmonoclonal antibodies 2H5 (BD Biosciences, Pharmiargen, San Diego,Calif.), 1M11, 3N10, 5J23, and 11K2.

FIG. 4 graphically depicts results of a chemotaxis assay whichdemonstrates that monocyte chemotaxis mediated by cytokines secretedfrom stimulated rheumatoid arthritis (RA) fibroblasts is inhibited bypan-MCP mAbs (1A1 and 11K2) and MCP-1 mAb D9.

FIGS. 5A, 5B, and 5C graphically depict results from a calcium fluxassay using 11K2 (mAb and Fab fragments thereof) at variousconcentrations, including none (FIG. 5A), 20 nM mAb (FIG. 5B), and 60 nMFab (FIG. 5C).

FIGS. 6A and 6B graphically depict results from a chemotaxis assay usingpan-MCP antibodies demonstrating pan-MCP antibodies 11K2 and 1A1increase MCP-2 mediated chemotaxis at low mAb concentrations (FIG. 6A).Blocking is also observed with MCP-2 mAb 281 (RD Systems, Minneapolis,MN). FIG. 6B graphically depicts a chemotaxis assay using the pan-MCPmAb 11K2 and the Fab fragment of 11K2.

FIGS. 7A, 7B, 7C, and 7D graphically depict results from a MCP-2 calciumflux assay which depicts results from 55.5 nM of MCP-2 alone, depictsresults which demonstrate that the 11K2 monoclonal antibody showsagonistic activity (FIG. 7B), and, as compared to 55.5 nM of MCP-2 alone(FIG. 7A), FIGS. 7C and 7D depict results which demonstrate that Fab andF(ab′)2 fragments of 11K2 are inhibitory in this assay.

FIG. 8 shows the amino acid and nucleotide sequences of the variableheavy region of the murine 1A1 antibody (FIG. 8A), as well as the aminoacid and nucleotide sequences of the 1A1 variable light region (FIG.8B). CDR regions are underlined.

FIG. 9 shows the amino acid and nucleotide sequences of murine 11K2variable heavy region (FIG. 9A), as well as the amino acid andnucleotide sequences of 11K2 variable light region (FIG. 9B). CDRregions are underlined.

DETAILED DESCRIPTION OF THE INVENTION

The reference works, patents, patent applications, and scientificliterature, inducting accession numbers to GenBank database sequences,that are referred to herein establish the knowledge of those with skillin the art are hereby incorporated by reference in their entirety to thesame extent as if each was specifically and individually indicated to beincorporated by reference. Any conflict between any reference citedherein and the specific teachings of this specification shall beresolved to favor of the latter.

Various definitions are made throughout this document. Most words havethe meaning that would be attributed to those words by one skilled inthe art. Words specifically defined either below or elsewhere in thisdocument have the meaning provided in the context of the presentinvention as a whole and as are typically understood by those skilled inthe art. Any conflict between an art-understood definition of a word orphrase and a definition of the word or phrase as specifically taught inthis specification shall be resolved in favor of the latter. Headingsused herein are for convenience and are not to be construed as limiting.

Standard reference works setting forth the general principles ofrecombinant DNA technology known to those of skill in the art includeAusubel et al., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley &Sons, New York, 1998; Sambrook et al., MOLECULAR CLONING: A LABORATORYMANUAL, 2d Ed., Cold Spring Harbor Laboratory Press, Plainview, N.Y.,1989; Kaufman et al., Eds., HANDBOOK OF MOLECULAR AND CELLULAR METHODSIN BIOLOGY AND MEDICINE, CRC Press, Boca Raton, 1995; McPherson, Ed.,DIRECTED MUTAGENESIS: A PRACTICAL APRROACH, IRL Press, Oxford, 1991.

As used herein, the term “antibody” is meant to refer to complete,intact antibodies, and Fab, Fab′, F(ab)₂, F_(v), and other fragmentsthereof. Complete, intact antibodies include, for example, monoclonalantibodies such as murine monoclonal antibodies, chimeric antibodies,anti-idiotypic antibodies, anti-anti-idiotypic antibodies, and humanizedantibodies.

As used herein, the term “binding” means the physical or chemicalinteraction between two proteins or compounds or associated proteins orcompounds or combinations thereof. Binding includes ionic, non-ionic,hydrogen bonds, Van der Waals, hydrophobic interactions, etc. Thephysical interaction, the binding, can be either direct or indirect,indirect being through or due to the effects of another protein orcompound. Direct binding refers to interactions that do not take placethrough or due to the effect of another protein or compound but insteadare without other substantial chemical intermediates.

As used herein, the term “isolated” nucleic acid molecule refers to anucleic acid molecule (DNA or RNA) that has been removed from its nativeenvironment. Examples of isolated nucleic acid molecules include, butare not limited to, recombinant DNA molecules contained in a vector,recombinant DNA molecules maintained in a heterologous host cell,partially or substantially purified nucleic acid molecules, andsynthetic DNA or RNA molecules. Preferably, an “isolated” nucleic acidis free of sequences which naturally flank the nucleic acid (i.e.,sequences located at the 5′ and 3′ ends of the nucleic acid) in thegenomic DNA of the organism from which the nucleic acid is derived. Forexample, in various embodiments, the isolated antibody-encoding nucleicacid molecule can contain less than about 50 kb, 25 kb, 5 kb, 4 kb, 3kb, 2 kb, 1 kb, 0.5 kb or 0.1 kb of nucleotide sequences which naturallyflank the nucleic acid molecule in genomic DNA of the cell from whichthe nucleic acid is derived. Moreover, an “isolated” nucleic acidmolecule, such as a cDNA molecule, can be substantially the of othercellular material or culture medium when produced by recombinanttechniques, or of chemical precursors or other chemicals when chemicallysynthesized.

As used herein, the term “isolated antibody” refers to an immunoglobulinmolecule or fragment thereof that has been removed from its nativeenvironment. This may include removing the antibody, or fragmentthereof, from ascites fluid, serum, blood, or tissue culture fluid, forexample.

As used herein, the term “β-chemokine” refers to a polypeptidecontaining four conserved cysteine residues characteristic ofβ-chemokines (e.g., as described in Van Coillie et al. (1999) Cytokine &Growth Factor Rev. 10:61-86), wherein the first two conserved cysteinesare adjacent.

As used herein, the term “inhibiting the activity of β-chemokines”refers to causing a decrease in the relative activity of β-chemokines(e.g. MCP-1, MCP-2, mCP-3) in the presence of the antibody orantigen-binding fragment thereof in comparison with the activityobserved in the absence of the antibody or antigen-binding fragmentthereof.

As used herein, the term “sign of an inflammatory disorder” refers toobservable or measurable indications of pathological inflammation,including, but not limited to edema, fever, emigration of leukocytes,proliferation of blood vessels, proliferation of connective tissue,redness, localized heat, exudation, and other signs as described inROBBINS PATHOLOGIC BASIS OF DISEASE, 4^(TH) EDITION, R. S. Cotran etal., Eds. W.B. Saunders, Co., 1989.

As used herein, the term “blocking chemotaxis” refers to a decrease inthe relative amount of chemotactic activity of cells in the presence ofthe antibody or antigen-binding fragment thereof in comparison withchemotactic activity observed in the absence of the antibody orantigen-binding fragment thereof.

As used herein, the term “MCP MRHAS Motif” refers to an amino acid motifin human MCP-1 and MCP-3 termed Meningitis Related Homologous AntigenicSequence. For human MCP-1, the MRHAS amino acid motif isGln-Thr-Gln-Thr-Pro-Lys-Thr (SEQ ID NO:1); and for human MCP-3, theMRHAS motif is Lys-Thr-Gln-Thr-Pro-Lys-Leu (SEQ ID NO:2).

As used herein “conservative change” refers to alterations that aresubstantially conformationally or antigenically neutral; producingminimal changes in the tertiary structure of the mutant polypeptides, orproducing minimal changes in the antigenic determinants of the mutantpolypeptides, respectively, as compared to the native protein. Suchconservative changes, when referring to chemokines refer to amino acidsubstitutions that do not ablate the activity of the subject chemokine,as measured by its ability to perform at least one function of thechemokine such as, but not limited to inducing chemotaxis, inducingenzyme or cytokine production, or binding to its receptor. Whenreferring to the antibodies and antibody fragments of the invention, aconservative change means an amino acid substitution that does notrender the antibody incapable of binding to the subject chemokine(s).

Those of ordinary skill in the art will be able to predict which aminoacid substitutions can be made while maintaining a high probability ofbeing conformationally and antigenically neutral. Such guidance isprovided, for example in Berzofsky, (1985) Science 229:932-940 and Bowieet al. (1990) Science 247:1306-1310. Factors to be considered thataffect the probability of maintaining conformational and antigenicneutrality include, but are not limited to (a) substitution ofhydrophobic amino acids is less likely to affect antigenicity becausehydrophobic residues are more likely to be located in a protein'sinterior, (b) substitution of physiochemically similar, amino acids isless likely to affect conformation because the substituted amino acidstructurally mimics the native amino acid; and (c) alteration ofevolutionary conserved sequences is likely adversely affect conformationas such conservation suggests that the amino acid sequences may havefunctional importance.

One of ordinary skill in the art will be able to assess alterations inprotein conformation using well-known assays, such as, but not limitedto microcomplement fixation methods (Wasserman et al. (1961) J. Immunol.87:290-295; Levine et al. (1967) Meth. Enzymol. 11:928-936) and throughbinding studies using conformation-dependent monoclonal antibodies(Lewis et al. (1983) Biochem. 22:948-954).

As used herein, “therapeutic composition” refers to a composition whichdirectly or indirectly ameliorates a disease condition. That is,administration of the composition alleviates at least one symptom of adisease or disorder.

As used herein, “immunoaffinity resin” refers to a solid substrate towhich at least one antibody is bound via a region other than its antigencombining site, thereby allowing the bound antibody to bind to itsepitope. Many resins are known in the art and are routinely used to formimmunoaffinity resins. Any such resin may be used to form theimmunoaffinity resins of the invention.

Antibodies and Antibody Fragments

The invention provides antibodies (e.g., monoclonal and polyclonalantibodies, single chain antibodies, chimeric antibodies,bifunctional/bispecific antibodies, humanized antibodies, humanantibodies, and complementary determining region (CDR)-graftedantibodies), including compounds which include CDR sequences whichspecifically recognize a plurality of β-chemokines, particularlymonocyte chemotactic proteins (e.g., MCP-1, MCP-2, and MCP-3) orfragments thereof.

Antibody fragments, including Fab, Fab′, F(ab′)₂, and F_(v), are alsoprovided by the invention. The term “specific for,” when used todescribe antibodies of the invention, indicates that the antibodyexhibits appreciable affinity for antigen or a preferred epitope, i.e.plurality of β-chemokines, and, preferably, does not exhibitcrossreactivity (i.e., are able to distinguish β-chemokines from otherpolypeptides by virtue of measurable differences in binding affinity,despite the possible existence of localized sequence identity, homology,or similarity between β-chemokines and such polypeptides).

It will be understood that specific antibodies may also interact withother proteins (for example, Staphylococcus aureus protein A or otherantibodies in ELISA techniques) through interactions with sequencesoutside the variable region of the antibodies, and, in particular, inthe constant region of the molecule. Screening assays to determinebinding specificity of an antibody of the invention are well known androutinely practiced in the art. For a comprehensive discussion of suchassays, see Harlow et al. (Eds.), ANTIBODIES: A LABORATORY MANUAL; ColdSpring Harbor Laboratory; Cold Spring Harbor, N.Y., 1988, Chapter 6.Antibodies that recognize and bind fragments of the β-chemokines arealso contemplated, provided that the antibodies are specific forβ-chemokines. Antibodies of the invention can be produced using anymethod well known and routinely practiced in the art.

For the production of polyclonal antibodies, various suitable hostanimals (e.g., rabbit, goat, mouse or other mammal) may be immunized byinjection with the native protein, or a synthetic variant thereof, or aderivative of a β-chemokine or combination of more than one β-chemokine.For example, but not by way of limitation, a cocktail of MCP-1, MCP-2and MCP-3 may be used as an immunogen to induce an immune responseagainst these β-chemokines.

An appropriate immunogenic preparation can contain, for example, apreparation of isolated, native β-chemokines, recombinantly expressedmonocyte chemotactic proteins or chemically synthesized β-chemokines.The preparation can further include an adjuvant. Various adjuvants usedto increase the immunological response include, but are not limited to,Freund's (complete and incomplete), mineral gels (e.g., aluminumhydroxide), surface active substances (e.g., lysolecithin, pluronicpolyols, polyanions, peptides, oil emulsions, dinitrophenol, etc.),human adjuvants such as Bacille Calmette-Guerin and Corynebacteriumparvum, or similar immunostimulatory agents. If desired, the antibodymolecules directed against the β-chemokines can be isolated from themammal (e.g., from the blood) and further purified by well-knowntechniques, such as protein A chromatography to obtain theimmunoglobulin fraction.

The term “monoclonal antibody” or “monoclonal antibody composition,” asused herein, refers to a population of antibody molecules that containonly one species of an antigen-binding site capable of immunoreactingwith a particular epitope of a β-chemokine. A monoclonal antibodycomposition thus typically displays a single binding affinity for aparticular epitope of a β-chemokine with which it immunoreacts. Thepan-antibodies of the invention specifically recognize and specificallybind to more than one type of β-chemokine. For preparation of monoclonalantibodies directed towards a particular β-chemokine, or derivatives,fragments, analogs or homologs thereof, any technique that provides forthe production of antibody molecules by continuous cell line culture maybe utilized. Such techniques include, but are not limited to, thehybridoma technique (see Kohler & Milstein (1975) Nature 256:495-497);the trioma technique; the human B-cell hybridoma technique (see Kozbor,et al. (1983) Immunol. Today 4:72) and the EBV hybridoma technique toproduce human monoclonal antibodies (see Cole, et al., 1985 In:MONOCLONAL ANTIBODIES AND CANCER THERAPY, Alan R. Liss, Inc., pp.77-96). Human monoclonal antibodies may be utilized in the practice ofthe present invention and may be produced by using human hybridomas (seeCote, et al., 1983. Proc. Natl. Acad. Sci. USA 80:2026-2030) or bytransforming human B-cells with Epstein Barr Virus in vitro (see Cole,et al. (1985) In: MONOCLONAL ANTIBODIES AND CANCER THERAPY, Alan R.Liss, Inc., pp. 77-96).

In certain embodiments the antibodies, and fragments thereof, bind toregions in the β-chemokines (e.g., MCP-1, MCP-2 and MCP-3). In someembodiments, the antibodies or antigen-binding fragments thereof bindMCP-2 and at least one other β-chemokine (e.g., MCP-1 or MCP-3). Thus,in some embodiments, the antibodies or fragments thereof bind MCP-1 andMCP-2 and in other embodiments the antibodies or fragments thereof bindMCP2 and MCP-3. In other embodiments, the antibodies or antigen-bindingfragments thereof bind MCP-1, MCP-2 and MCP-3. In other embodiments, theantibodies or antigen-binding fragments thereof bind MCP-1 and MCP-3other than to regions containing the MRHAS motifs, QTQTPKT (MCP-1) andKTQTPKL (MCP-3).

In certain embodiments, the antibodies or antigen-binding fragmentsthereof comprise antibodies selected from the group consisting of 11K2.1(ATCC Accession No. PTA-3987), 6D21.1 (ATCC Accession No. PTA-3989),4N4.1 (ATCC Accession No. PTA-3994). 5A13.1 (ATCC Accession No.PTA-3995), 7H1.1 (ATCC Accession No. PTA-3985), 1A1.1 (ATCC AccessionNo. PTA-3990), 6I5.1 (ATCC Accession No. PTA-3986), 2O24.1 (ATCCAccession No. PTA-3993), 9B11.1 (ATCC Accession No. PTA-3992), 9B12.1(ATCC Accession No. PTA-3996), 9C11.1 (ATCC Accession No. PTA-3988), and12F15.1 (ATCC Accession No. PTA-3991), or antigen-binding fragments ofthese antibodies.

The amino acid and nucleotide sequences of the light and heavy variableregions of antibody 1A1 are shown in FIG. 8, where the CDRs areunderlined. The amino acid sequence of the 1A1 heavy chain variableregion is set forth as SEQ ID NO:11 and the light chain variable regionof 1A1 is set forth as SEQ ID NO:12. Furthermore, the CDRs of antibody1A1 are described as SEQ ID NOs:13-15 (heavy chain variable region) andSEQ ID NOs:16-18 (light chain variable region).

In one embodiment, the Invention features an antibody, orantigen-binding fragment comprising the variable heavy chain regiondescribed in SEQ ID NO:11 and the variable light chain region describedin SEQ ID NO:12. In another embodiment, the invention features anantibody or antigen-binding fragment comprising CDRs from the 1A1 heavychain variable region as described in SEQ ID NO:11, and CDRs from the1A1 light chain variable region as described in SEQ ID NO:12.

The amino acid and nucleotide sequences of the light and heavy variableregions of antibody 11K2 are shown in FIG. 9, where the CDRs areunderlined. The amino acid sequence of the 11K2 heavy chain variableregion is set forth as SEQ ID NO:27 and the light chain variable regionof 11K2 is set forth as SEQ ID NO:28. In addition, the CDRs of antibody11K2 are described as SEQ ID NOs:29-31 (heavy chain variable region) andSEQ ID NOs:32-34 (light chain variable region).

In one embodiment, the invention features an antibody, orantigen-binding fragment comprising the variable heavy chain regiondescribed in SEQ ID NO:27 and the variable light chain region describedin SEQ ID NO:28. In another embodiment, the invention features anantibody or antigen-binding fragment comprising CDRs from the 11K2 heavychain variable region as described in SEQ ID NO:27, and CDRs from the1A1 light chain variable region as described is SEQ ID NO:28.

In producing the antibodies of the invention, the immunogens may be apreparation containing at least one β-chemokine, preferably more thanone β-chemokine. In some embodiments, the β-chemokines are humanmonocyte chemotactic proteins (MCPs), including MCP-1, MCP-2 and MCP-3.The β-chemokines may be native or recombinantly produced β-chemokines.In some embodiments, the immunogens may be antigenic fragments ofβ-chemokines, such as fragments of MCPs which may optionally beconjugated to a carrier molecule to impart a stronger immune responseupon administration to an animal. The β-chemokine immunogens, such asMCPs may contain additions, deletions and/or substitutions of aminoacids, provided that the alterations do not ablate antigenicity of themutated β-chemokines such that antibodies against the mutant versions donot bind native β-chemokines. Preferably, the amino acid substitutionsare conservative changes in the amino acid sequence, provided the MCPmolecules remain antigenic.

The sequences of MCP proteins and polynucleotides encoding such proteinsare known and may be found, for example, in publicly available sequencedatabases such as GenBank. In addition, the sequences of various MCPshave been published, and may be found, for example, in Furutani et al.(1989) Biochem. Biophys. Res. Commun. 159:249-235, Yoshimura et al.(1989) FEBS Lett. 244:487-493 (MCP-1); Van Coillie et al. (1997)Biochem. Biophys. Res. Commun. 231:726-730, Van Coillie et al. (1997)Genomics 21:323-331 (MCP-2); Opdenakker et al. (1993) Biochem Biophys.Res. Commun. 191:535-542, Opdenakker et al. (1993) Genomics 21:403-408(MCP-3), the disclosure of each of which is incorporated by referenceherein in its entirety.

The antibodies of the invention may be any isotype, including IgA, IgD,IgE, IgG1, IgG2, IgG2a, IgG2b, IgG3, or IgM. The isotypes may be changedby isotype switching techniques as is known in the art or may begenetically engineered by CDR grafting, chimeric antibody formation orhumanization.

According to the invention, techniques can be adapted for the productionof single-chain antibodies specific to β-chemokines (see e.g., U.S. Pat.No. 4,946,778). In addition, methods can be adapted for the constructionof Fab expression libraries (see e.g., Huse, et al. (1989) Science246:1275-1281) to allow rapid and effective identification of monoclonalFab fragments with the desired specificity for β-chemokines orderivatives, fragments, analogs or homologs thereof. Non-humanantibodies can be “humanized” by techniques well known in the art (seee.g., U.S. Pat. No. 5,225,539). In one method, the non-human CDRs areinserted into a human antibody or consensus antibody framework sequence.Further changes can then be introduced into the antibody framework tomodulate affinity or immunogenicity. Antibody fragments that contain theidiotypes to β-chemokines may be produced by techniques known in the artincluding, but not limited to: (i) an F(ab′)₂ fragment produced bypepsin digestion of an antibody molecule; (ii) an Fab fragment generatedby reducing the disulfide bridges of an F(ab′)₂ fragment; (iii) an Fabfragment generated by the treatment of the antibody molecule with papainand a reducing agent and (iv) F_(v) fragments.

Additionally, recombinant anti-β-chemokine antibodies, such as chimericand humanized monoclonal antibodies, comprising both humans andnon-human portions, which can be made using standard recombinant DNAtechniques, are within the scope of the invention. Chimeric and/orhumanized antibodies have the same or similar binding specificity andaffinity as a mouse or other nonhuman antibody that provides thestarting material for construction of a chimeric or humanized antibody.

“Chimeric antibodies” refers to antibodies wherein one portion of eachof the amino acid sequences of heavy and light chains is homologous tocorresponding sequences is antibodies derived from a particular speciesor belonging to a particular class, while the remaining segment of thechains is homologous to corresponding sequences from another species. Inone embodiment, the invention features a chimeric antibody orantigen-binding fragment, in which the variable regions of both lightand heavy chains mimics the variable regions of antibodies derived fromone species of mammals, while the constant portions are homologous tothe sequences in antibodies derived from another species. In a preferredembodiment of the invention, chimeric antibodies are made by graftingCDRs from a mouse antibody onto the framework regions of a humanantibody.

“Humanized antibodies” refer to antibodies which comprise at least onechain comprising variable region framework residues substantially from ahuman antibody chain (referred to as the acceptor immunoglobulin orantibody) and at least one complementarity determining region (CDR)substantially from a non-human-antibody (e.g., mouse). In addition tothe grafting of the CDRs, humanized antibodies typically undergo furtheralterations in order to improve affinity and/or immmunogenicity.

Such chimeric and humanized monoclonal antibodies can be produced byrecombinant DNA techniques known in the art, for example using methodsdescribed in PCT International Application No. PCT/US86/02296; EuropeanPatent Application No. 184,187; European Patent Application No. 171,496;European Patent Application No. 173,494; PCT International PublicationNo. WO 86/01533; U.S. Pat. No. 4,816,567; European Patent ApplicationNo. 125,023; Better et al. (1988) Science 240:1041-1043; Liu et al.(1987) Proc. Natl. Acad. Sci. USA 84:3439-3443; Liu et al. (1987) J.Immunol. 139:3521-3526; Sun et al. (1987) Proc. Nat. Acad. Sci. USA84:214-218; Nishimura et al. (1987) Cancer Res. 47:999-1005; Wood et al.(1985) Nature 314:446-449; Shaw et al. (1988) J. Natl. Cancer Inst.80:1553-1559); Morrison (1985) Science 229:1202-1207; Oi et al. (1986)BioTechniques 4:214; U.S. Pat. No. 5,225,539; Jones et al. (1986) Nature321:552-525; Verhoeyan et al. (1988). Science 239:1534; and Beidler etal. (1988) J. Immunol. 141:4053-4060, Queen et al., Proc. Natl. Acad.Sci. USA 86:10029-10033 (1989), U.S. Pat. Nos. 5,530,101, 5,585,089,5,693,761, 5,693,762, Selick et al., WO 90/07861, and Winter, U.S. Pat.No. 5,225,539.

The CDRs of the 1A1 antibody can be used to produce humanized andchimeric antibodies. In one embodiment, the invention provides anisolated antibody, or antigen-binding fragment thereof, which binds aplurality of β-chemokines, wherein said β-chemokines comprise MCP-1 andat least one other β-chemokine (e.g., MCP-2, MCP-3), and wherein theantibody or antigen-binding fragment comprises at least one of thefollowing CDRs: CDR1, CDR2, or CDR3, from the 1A1 heavy chain variableregion set forth as SEQ ID NO:11. In another embodiment, the inventionprovides an isolated antibody, or antigen-binding fragment thereof,which binds a plurality of β-chemokines, wherein said β-chemokinescomprise MCP-1 and at least one other β-chemokine (e.g. MCP-2, MCP-3),which comprises at least one of the following CDR combinations: CDR1 andCDR2; CDR1 and CDR3; CDR2 and CDR3; and CDR1, CDR2, and CDR3, from the1A1 heavy chain variable region described in SEQ ID NO:11.

In one embodiment, the antibody of the invention is a chimeric antibody.In another embodiment, the antibody of the invention is a humanizedantibody.

The invention also provides an isolated antibody, or antigen-bindingfragment thereof, which binds a plurality of β-chemokines, wherein saidβ-chemokines comprise MCP-1 and at least one other β-chemokine (e.g.MCP-2, MCP-3), wherein the antibody or antigen-binding fragmentcomprises at least one of the following CDRs: CDR1, CDR2, or CDR3, fromthe 1A1 light chain variable region described in SEQ ID NO:12. Inanother embodiment, the invention provides an isolated antibody, orantigen-binding fragment thereof, which binds a plurality ofβ-chemokines, wherein said β-chemokines comprise MCP-1 and at least oneother β-chemokine (e.g. MCP-2, MCP-3), wherein the antibody orantigen-binding fragment comprises at least one of the following CDRcombinations: CDR1 and CDR2; CDR1 and CDR3; CDR2 and CDR3; or CDR1,CDR2, and CDR3, from the 1A1 light chain variable region described inSEQ ID NO:12. In one embodiment, the antibody of the invention is achimeric antibody. In another embodiment, the antibody of the inventionis a humanized antibody.

In one embodiment, the invention provides an antibody or antigen-bindingfragment which binds a plurality of β-chemokines, wherein saidβ-chemokines comprise MCP-1 and at least one other β-chemokine (e.g.,MCP-2, MCP-3), and wherein said antibody or antigen-binding fragmentcomprises a heavy chain variable region having a CDR1 domain comprisingthe sequence set forth as SEQ ID NO:13, a CDR2 domain comprising thesequence set forth as SEQ ID NO:14, and a CDR3 domain comprising thesequence set forth as SEQ ID NO:15. In another embodiment, the inventionfeatures an antibody or antigen-binding fragment which binds a pluralityof β-chemokines, wherein said β-chemokines comprise MCP-1 and at leastone other β-chemokine (e.g., MCP-2, MCP-3), and wherein said antibody orantigen-binding fragment comprises a light chain variable region havinga CDR1 domain comprising the sequence set forth as SEQ ID NO:16 a CDR2domain comprising the sequence set forth as SEQ ID NO:17, and a CDR3domain comprising the sequence set forth as SEQ ID NO:18. In anotherembodiment, the CDR domains of the 1A1 antibody, set forth as SEQ IDNOs:13-18, are used to make a chimeric antibody. In yet anotherembodiment, the CDR domains of the 1A1 antibody, set forth as SEQ IDNOs:13-18, are used to make a humanized antibody.

The CDRs of the 11K2 antibody can be also used to produce humanized andchimeric antibodies. The invention features an isolated antibody orantigen-binding fragment thereof which binds a plurality ofβ-chemokines, wherein said β-chemokines comprise MCP-1 and at least oneother β-chemokine (e.g., MCP-2, MCP-3), and wherein said antibody orantigen-binding fragment comprises at least one of the following CDRs:CDR1, CDR2, or CDR3, from the 11K2 heavy chain variable region set forthas SEQ ID NO:27. In an additional embodiment, the invention features anisolated antibody or antigen-binding fragment thereof which binds aplurality of β-chemokines, wherein said β-chemokines comprise MCP-1 andat least one other β-chemokine (e.g., MCP-2, MCP-3), and wherein saidantibody or antigen-binding fragment comprises at least one of thefollowing CDR combinations: CDR1 and CDR2; CDR1 and CDR3; CDR2 and CDR3;or CDR1, CDR2, and CDR3, from the 11K2 heavy chain variable regiondescribed is SEQ ID NO:27. In one embodiment, the antibody of theinvention is a chimeric antibody. In another embodiment, the antibody ofthe invention is a humanized antibody.

The invention also provides an isolated antibody, or antigen-bindingfragment thereof which binds a plurality of β-chemokines, wherein saidβ-chemokines comprise MCP-1 and at least one other β-chemokine (e.g.MCP-2, MCP-3), and wherein said antibody or antigen-binding fragmentcomprises at least one of the following CDRs: CDR1, CDR2, or CDR3, fromthe 11K2 light chain variable region set forth as SEQ ID NO:28. Inanother embodiment, the invention provides an isolated antibody, orantigen-binding fragment thereof which binds a plurality ofβ-chemokines, wherein said β-chemokines comprise MCP-1 and at least oneother β-chemokine (e.g. MCP-2, MCP-3), and wherein said antibody orantigen-binding fragment comprises at least one of the following CDRcombinations: CDR1 and CDR2; CDR1 and CDR3; CDR2 and CDR3; or CDR1,CDR2, and CDR3, from the 11K2 light chain variable region described inSEQ ID NO:28. In one embodiment, the antibody of the invention is achimeric antibody. In another embodiment, the antibody of the inventionis a humanized antibody.

In another embodiment, the invention features an antibody orantigen-binding fragment thereof which binds a plurality ofβ-chemokines, wherein said β-chemokines comprise MCP-1 and at least oneother β-chemokine (e.g., MCP-2, MCP-3), and wherein said antibody orantigen-binding fragment comprises a heavy chain variable region havinga CDR1 domain comprising the sequence set forth as SEQ ID NO:29, a CDR2domain comprising the sequence set forth as SEQ ID NO:30, and a CDR3domain comprising the sequence set forth as SEQ ID NO:31. In anotherembodiment, the invention features an antibody or antigen-bindingfragment thereof which binds a plurality of β-chemokines, wherein saidβ-chemokines comprise MCP-1 and at least one other β-chemokine (e.g.,MCP-2, MCP-3), and wherein said antibody or antigen-binding fragmentcomprises a light chain variable region having a CDR1 domain comprisingthe sequence set forth as SEQ ID NO:32, a CDR2 domain comprising thesequence set forth as SEQ ID NO:33, and a CDR3 domain comprising thesequence set forth as SEQ ID NO:34. In another embodiment, the CDRdomains of the 11K2 antibody, as described in SEQ ID NOs:29-34, are usedto make a chimeric antibody. In yet another embodiment, the CDR domainsof the 11K2 antibody, as described in SEQ ID NOs:29-34, are used to makea humanized antibody.

In general, humanized antibodies are produced by substituting mouse CDRsinto a human variable domain framework which is most likely to result inretention of the correct spatial orientation of the CDRs if the humanvariable domain framework adopts the same or similar conformation to themouse variable framework from which the CDRs originated. This isachieved by obtaining the human variable domains from human antibodieswhose framework sequences exhibit a high degree of sequence identitywith the murine variable framework domains from which the CDRs werederived. The heavy and light chain variable framework regions can bederived from the same or different human antibody sequences. The humanantibody sequences can be the sequences of naturally occurring humanantibodies or can be consensus sequences of several human antibodies.See Kettleborough et al., Protein Engineering 4:773 (1991); Kolbinger etal., Protein Engineering 6:971 (1993) and Carter et al., WO 92/22653.

Having identified the complementarity determining regions of the murinedonor immunoglobulin and appropriate human acceptor immunoglobulins, thenext step is to determine which, if any, residues from these componentsshould be substituted to optimize the properties of the resultinghumanized antibody. In general, substitution of human amino acidresidues with murine should be minimized, because introduction of murineresidues increases the risk of the antibody eliciting ahuman-anti-mouse-antibody (HAMA) response in humans.

Certain amino acids from the human variable region framework residuesare selected for substitution based on their possible influence on CDRconformation and/or binding to antigen. The unnatural juxtaposition ofmurine CDR regions with human variable framework region can result inunnatural conformational restraints, which, unless corrected bysubstitution of certain amino acid residues, lead to loss of bindingaffinity.

The selection of amino acid residues for substitution is determined, inpart, by computer modeling. In general, molecular models are producedstarting from solved structures for immunoglobulin chains or domainsthereof. The chains to be modeled are compared for amino acid sequencesimilarity with chains or domains of solved three-dimensionalstructures, and the chains or domains showing the greatest sequencesimilarity is/are selected as starting points for construction of themolecular model. Chains or domains sharing at least 50% sequenceidentity are selected for modeling, and preferably those sharing atleast 60%, 70%, 80%, 90% sequence identity or more are selected formodeling. The solved starting structures are modified to allow fordifferences between the actual amino acids in the immunoglobulin chainsor domains being modeled, and those in the starting structure. Themodified structures are then assembled into a composite immunoglobulin.Finally, the model is refined by energy minimization and by verifyingthat all atoms are within appropriate distances from one another andthat bond lengths and angles are within chemically acceptable limits.

The selection of amino acid residues for substitution can also bedetermined, in part, by examination of the characteristics of the aminoacids at particular locations, or empirical observation of the effectsof substitution or mutagenesis of particular amino acids. For example,when an amino acid differs between a murine variable region frameworkresidue and a selected human variable region framework residue, thehuman framework amino acid should usually be substituted by theequivalent framework amino acid from the mouse antibody when it isreasonably expected that the amino acid:

noncovalently binds antigen directly,

is adjacent to a CDR region,

otherwise interacts with a CDR region (e.g., is within about 3-6 Å of aCDR region as determined by computer modeling), or

participates in the VL-VH interface.

Residues which “noncovalently bind antigen directly” include amino acidsin positions in framework regions which are have a good probability ofdirectly interacting with amino acids on the antigen according toestablished chemical forces, for example, by hydrogen bonding, Van derWaals forces, hydrophobic interactions, and the like.

Residues which are “adjacent to a CDR region” include amino acidresidues in positions immediately adjacent to one or more of the CDRs inthe primary sequence of the humanized immunoglobulin chain, for example,in positions immediately adjacent to a CDR as defined by Kabat, or a CDRas defined by Chothia (See e.g., Chothia and Lesk J M B 196:901 (1987)).These amino acids are particularly likely to interact with the aminoacids in the CDRs and, if chosen from the acceptor, to distort the donorCDRs and reduce affinity. Moreover, the adjacent amino acids mayinteract directly with the antigen (Amit et al., Science, 233:747(1986), which is incorporated herein by reference) and selecting theseamino acids from the donor may be desirable to keep all the antigencontacts that provide affinity in the original antibody.

Residues that “otherwise interact with a CDR region” include those thatare determined by secondary structural analysis to be in a spatialorientation sufficient to effect a CDR region. In one embodiment,residues that “otherwise interact with a CDR region” are identified byanalyzing a three-dimensional model of the donor immunoglobulin (e.g., acomputer-generated model). A three-dimensional model, typically of theoriginal donor antibody, shows that certain amino acids outside of theCDRs are close to the CDRs and have a good probability of interactingwith amino acids in the CDRs by hydrogen bonding, Van der Waals forces,hydrophobic interactions, etc. At those amino acid positions, the donorimmunoglobulin amino acid rather than the acceptor immunoglobulin aminoacid may be selected. Amino acids according to this criterion willgenerally have a side chain atom within about 3 angstrom units (Å) ofsome atom in the CDRs and must contain an atom that could interact withthe CDR atoms according to established chemical forces, such as thoselisted above.

Amino acids that are capable of interacting with amino acids in theCDRs, may be identified in yet another way. The solvent accessiblesurface area of each framework amino acid is calculated in two ways: (1)in the intact antibody, and (2) in a hypothetical molecule consisting ofthe antibody with its CDRs removed. A significant difference betweenthese numbers of about 10 square angstroms or more shows that access ofthe framework amino acid to solvent is at least partly blocked by theCDRs, and therefore that the amino acid is making contact with the CDRs.Solvent accessible surface area of an amino acid may be calculated basedon a three-dimensional model of an antibody, using algorithms known inthe art (e.g., Connolly, J. Appl. Cryst. 16:548 (1983) and Lee andRichards, J. Mol. Biol. 55:379 (1971), both of which are incorporatedherein by reference). Framework amino acids may also occasionallyinteract with the CDRs indirectly, by affecting the conformation ofanother framework amino acid that in turn contacts the CDRs.

Residues which “participate in the VL-VH interface” or “packingresidues” include those residues at the interface between VL and VH asdefined, for example, by Novotny and Haber, Proc. Natl. Acad. Sci. USA,82:4592-66 (1985) or Chothia et al, supra. Generally, unusual packingresidues should be retained in the humanized antibody if they differfrom those in the human frameworks.

In general, one or more of the amino acids fulfilling the above criteriais substituted. In some embodiments, all or most of the amino acidsfulfilling the above criteria are substituted. Occasionally, there issome ambiguity about whether a particular amino acid meets the abovecriteria, and alternative variant immunoglobulins are produced, one ofwhich has that particular substitution, the other of which does not.Alternative variant immunoglobulins so produced can be tested in any ofthe assays described herein for the desired activity, and the preferredimmunoglobulin selected.

Usually the CDR regions is humanized antibodies are substantiallyidentical, and more usually, identical to the corresponding CDR regionsof the donor antibody. Although not usually desirable, it is sometimespossible to make one or more conservative amino acid substitutions ofCDR residues without appreciably affecting the binding affinity of theresulting humanized immunoglobulin. By conservative substitutions isintended combinations such as gly, ala; val, ile, leu; asp, glu; asn,gln; ser, thr; lys, arg; and phe, tyr.

Additional candidates for substitution are acceptor human frameworkamino acids that are unusual or “rare” for a human immunoglobulin atthat position. These amino acids can be substituted with amino acidsfrom the equivalent position of the mouse donor antibody or from theequivalent positions of more typical human immunoglobulins. For example,substitution may be desirable when the amino acid in a human frameworkregion of the acceptor immunoglobulin is rare for that position and thecorresponding amino acid in the donor immunoglobulin is common for thatposition in human immunoglobulin sequences; or when the amino acid inthe acceptor immunoglobulin is rare for that position and thecorresponding amino acid in the donor immunoglobulin is also rare,relative to other human sequences. These criterion help ensure that anatypical amino acid in the human framework does not disrupt the antibodystructure. Moreover, by replacing an unusual human acceptor amino acidwith an amino acid from the donor antibody that happens to be typicalfor human antibodies, the humanized antibody may be made lessimmunogenic.

The term “rare”, as used herein, indicates an amino acid occurring atthat position in less than about 20% but usually less than about 10% ofsequences in a representative sample of sequences, and the term“common”, as used herein, indicates an amino acid occurring in more thanabout 25% but usually more than about 50% of sequences in arepresentative sample. For example, all human light and heavy chainvariable region sequences are respectively grouped into “subgroups” ofsequences that are especially homologous to each other and have the sameamino acids at certain critical positions (Kabat et al., supra). Whendeciding whether as amino acid in a human acceptor sequence is “rare” or“common” among human sequences, it will often be preferable to consideronly those human sequences is the same subgroup as the acceptorsequence.

Additional candidates for substitution are acceptor human frameworkamino acids that would be identified as part of a CDR region under thealternative definition proposed by Chothia et al., supra. Additionalcandidates for substitution are acceptor human framework amino acidsthat would be identified as part of a CDR region under the AbM and/orcontact definitions.

Additional candidates for substitution are acceptor framework residuesthat correspond to a rare or unusual donor framework residue. Rare orunusual donor framework residues are those that are rare or unusual (asdefined herein) for murine antibodies at that position. For murineantibodies, the subgroup can be determined according to Kabat andresidue positions identified which differ from the consensus. Thesedonor specific differences may point to somatic mutations in the murinesequence which enhance activity. Unusual residues that are predicted toaffect binding are retained, whereas residues predicted to beunimportant for binding can be substituted.

Additional candidates for substitution are non-germline residuesoccurring in an acceptor framework region. For example, when an acceptorantibody chain (i.e., a human antibody chain sharing significantsequence identity with the donor antibody chain) is aligned to agermline antibody chain (likewise sharing significant sequence identitywith the donor chain), residues not snatching between acceptor chainframework and the germline chain framework can be substituted withcorresponding residues from the germline sequence.

Other than the specific amino acid substitutions discussed above, theframework regions of humanized immunoglobulins are usually substantiallyidentical, and more usually, identical to the framework regions of thehuman antibodies from which they were derived. Of course, many of theamino acids in the framework region make little or no directcontribution to the specificity or affinity of an antibody. Thus, manyindividual conservative substitutions of framework residues can betolerated without appreciable change of the specificity or affinity ofthe resulting humanized immunoglobulin. Thus, is one embodiment thevariable framework region of the humanized immunoglobulin shares atleast 85% sequence identity to a human variable framework regionsequence or consensus of such sequences. In another embodiment, thevariable framework region of the humanized immunoglobulin shares atleast 90%, preferably 95%, more preferably 96%, 97%, 98% or 99% sequenceidentity to a human variable framework region sequence or consensus ofsuch sequences. In general, however, such substitutions are undesirable.

In a preferred embodiment, humanized antibodies preferably exhibit aspecific binding affinity for antigen similar to that of the mouseantibody from which they were constructed. Usually the upper limit ofbinding affinity of the humanized antibodies for antigen is within afactor of three, four or five of that of the donor immunoglobulin. Oftenthe lower limit of binding affinity is also within a factor of three,four or five of that of donor immunoglobulin. Alternatively, the bindingaffinity can be compared to that of a humanized antibody having nosubstitutions (e.g., an antibody having donor CDRs and acceptor FRs, butno FR substitutions). In such instances, the binding of the optimizedantibody (with substitutions) is preferably at least two- to three-foldgreater, or three- to four-fold greater, than that of the unsubstitutedantibody. For making comparisons, activity of the various antibodies canbe determined, for example, by BIACORE (i.e., surface plasmon resonanceusing unlabelled reagents) or competitive binding assays.

In some embodiments, the chimeric proteins of the invention comprise anantigen-binding fragment of an antibody joined via peptide bonds to aheterologous protein. In such embodiments, the antigen-binding portionof the antibody binds MCP-2 and at least one other β-chemokine, such asMCP-1 or MCP-3. In some embodiments, the antigen-binding fragment of theantibody binds MCP-1, MCP-2 and MCP-3. In other embodiments, theantigen-binding fragment binds MCP-1 and MCP-3, but does not bind an MCPMRHAS motif.

In some embodiments, the heterologous proteins that are joined to theantigen-binding fragments of the antibodies of the invention areenzymes, toxins or a cytokine.

The invention also provides compositions comprising a chimeric proteinof the invention and at least one pharmaceutically acceptable carrier,as defined herein.

The invention also provides for antibodies, antigen-binding fragmentsand/or antibody fragments of the invention that have high bindingaffinity for β-chemokines. High binding affinity refers to bindingaffinities of, for example, about 10×10⁻¹² M (i.e. 10 pM) or less. Inone embodiment the antibodies, antigen-binding fragments and/or antibodyfragments have a Kd for binding to the β-chemokines (either binding aplurality of MCP's selected from MCP-1, MCP-2, and MCP-3 or that bindthe individual MCPs, i.e. an antibody or antigen-binding fragment thatbinds to MCP-1, MCP-2 or MCP-3) between about 10×10⁻¹² M (10 pM) andabout 8×10⁻¹² M (8 pM) including 9×10⁻¹ M (9 pM); alternatively betweenabout 9×10⁻¹² M (9 pM) and about 7×10⁻¹² M (7 pM) including 8×10⁻¹² M (8pM); alternatively between about 8×10⁻¹² M (8 pM) and about 6×10⁻¹² M (6pM) including 7×10⁻¹² M (7 pM); alternatively between about 7×10⁻¹² M (7pM) and about 5×10⁻¹² M (5 pM) including 6×10⁻¹² M (6 pM), alternativelybetween about 6×10⁻¹² M (6 pM) and about 4×10⁻¹² M (4 pM) including5×10⁻¹² M (5 pM), alternatively between about 5×10⁻¹² M (5 pM) and about3×10⁻¹² M (3 pM) including 4×10⁻¹² M (4 pM); alternatively between about4×10⁻¹² M (4 pM) and about 2×10⁻¹² M (2 pM) including 3×10⁻¹² M (3 pM);alternatively between about 3×10⁻¹² M (3 pM) and about 1×10⁻¹² M (1 pM)including 2×10⁻¹² M (2 pM); alternatively about 1×10⁻¹² M (1 pM) andabout 8×10⁻¹³ M (0.8 pM) including 9×10⁻¹³ M (0.9 pM); alternativelybetween about 9×10⁻¹³ M (0.9 pM) and about 7×10⁻¹³ M (0.7 pM) including8×10⁻¹³ M (0.8 pM); alternatively between about 8×10⁻¹³ M (0.8 pM) andabout 6×10⁻¹³ M (0.6 pM) including 7×10⁻¹³ M (0.7 pM); alternativelybetween about 7×10⁻¹³ M (0.7 pM) and about 5×10⁻¹³ M (0.5 pM) including6×10⁻¹³ M (0.6 pM), alternatively between about 6×10⁻¹³ M (0.6 pM) andabout 4×10⁻¹³ M (0.4 pM) including 5×10⁻¹³ M (0.5 pM), alternativelybetween about 5×10⁻¹³ M (0.5 pM) and about 3×10⁻¹³ M (0.3 pM) including4×10⁻¹³ M (0.4 pM); alternatively between about 4×10⁻¹³ M (0.4 pM) andabout 2×10⁻¹³ M (0.2 pM) including 3×10⁻¹³ M (0.3 pM); alternativelybetween about 3×10⁻¹³ M (0.3 pM) and about 1×10⁻¹³ M (0.1 pM) including2×10⁻¹³ M (0.2 pM). The invention would include for example an antibodyor antigen-binding fragment thereof that binds to MCP-1, MCP-2 or MCP-3wherein the antibody or antigen-binding fragment thereof has a Kd forbinding to MCP-1, MCP-2 or MCP-3 selected from the following Kd's: about10×10⁻¹³ M (1 pM), 9×10⁻¹³ M (0.9 pM), 8×10⁻¹³ M (0.8 pM), 7×10⁻¹³ M(0.7 pM), 6×10⁻¹³ M (0.6 pM), 5×10⁻¹³ M (0.5 pM), 4×10⁻¹³ M (0.4 pM),3×10⁻¹³ M (0.3 pM), 2×10⁻¹³ M (0.2 pM) or 1×10⁻¹³ M (0.1 pM). (Anexample of such an antibody would include for example 11K2 in which theantibody has a binding affinity for human MCP-1 of about 0.4 pM.) Theinvention would also include for example an antibody or antigen-bindingfragment thereof that binds to a plurality of MCP's (i.e. MCP-1 andMCP-2 or MCP-1 and MCP-3 or MCP-1, MCP-2 and MCP-3 or MCP-2 and MCP-3)wherein the antibody or antigen-binding fragment thereof has a Kd forbinding to at least one of the MCP's (i.e. MCP-1, MCP-2 or MCP-3)selected from the following Kd's: about 10×10⁻¹³ M (1 pM), 9×10⁻¹³ M(0.9 pM), 8×10⁻¹³ M (0.8 pM), 7×10⁻¹³ M (0.7 pM), 6×10⁻¹³ M (0.6 pM),5×10⁻¹³ M (0.5 pM), 4×10⁻¹³ M (0.4 pM), 3×10⁻¹³ M (0.3 pM), 2×10⁻¹³ M(0.2 pM) or 1×10⁻¹³ M (0.1 pM). (An example of such an antibody wouldalso include for example 11K2 in which the antibody has a bindingaffinity for human MCP-1 of about 0.4 pM. and also binds MCP-2 andMCP-3). Methods for measuring the binding affinity of the antibody,antigen-binding fragment and or antibody fragment for the variousβ-chemokine(s) are known to those of skill in the art and include, forexample, the kinetic exclusion assay illustrated in Example 4 herewith.

The invention also provides for anybodies, antigen-binding fragmentsand/or antibody fragments comprising a Fab fragment wherein the Fabfragment has a Kd for binding MCP-1, MCP-2 or MCP-3 of, for example,about 1.5×10⁻¹¹ M (i.e. 15 pM) or less. The invention would include forexample an antibody, antigen-binding fragment and/or antibody fragmentthereof wherein the Fab fragment has a Kd for binding to MCP-1, MCP-2 orMCP-3 selected from the following Kd's: about 1.8×10⁻¹¹ M (18 pM), about1.7×10⁻¹¹ M (17 pM), about 1.6×10⁻¹¹ M (15 pM), about 1.5×10⁻¹¹ M (15pM), 1.4×10⁻¹¹ M (14 pM), 1.3×10⁻¹¹ M (13 pM), 1.2×10⁻¹¹ M (12 pM),1.1×10⁻¹¹ M (11 pM), 1×10⁻¹¹ M (10 pM), 0.9×10⁻¹¹ M (9 pM), 0.8×10⁻¹¹ M(8 pM), 0.7×10⁻¹¹ M (7 pM), 0.6×10⁻¹¹ M (6 pM), 0.5×10⁻¹¹ M (5 pM),0.4×10⁻¹¹ M (4 pM), 0.3×10⁻¹¹ M (3 pM), 0.2×10⁻¹¹ M (2 pM) or 0.1×10⁻¹¹M (1 pM). Methods for measuring the binding affinity of the antibody,antigen-binding fragment and or antibody fragment are known to those ofskill in the art and include, for example, the kinetic exclusion assayillustrated in Example 4 herewith.

The invention also provides for antibodies, antigen-binding fragmentsand/or antibody fragments that have the following binding affinity forthe β-chemokines (either binding a plurality of MCP's selected fromMCP-1, MCP-2, and MCP-3 or that bind the individual MCPs, i.e. anantibody or antigen-binding fragment that binds to MCP-1, MCP-2 orMCP-3). In one embodiment the binding affinity is between about 5×10⁻⁸ Mand about 5×10⁻¹² M, in some embodiments the binding affinity is about5×10⁻⁹ M to about 5×10⁻¹¹ M, in some embodiments the binding affinity isabout 5×10⁻⁷ M to about 5×10⁻⁸ M, in some embodiments the bindingaffinity is about 5×10⁻⁸ M to about 5×10⁻⁹ M, in some embodiments thebinding affinity is about 5×10⁻⁹ M to about 5×10⁻¹⁰ M, in someembodiments the binding affinity is about 5×10⁻¹⁰ M to about 5×10⁻¹¹ M.

The antibodies and antibody fragments of the invention can out-competefor the binding of ligand than other, known anti -β-chemokine antibodieswith an IC₅₀ of less than about 10 μg/ml, more preferably less thanabout 5 μg/ml, more preferably less than about 4 μg/ml, more preferablyless than about 3 μg/ml, more preferably less than about 2 μg/ml, andmore preferably less than about 1.0 μg/ml.

In one embodiment, methods for the screening of antibodies that possessthe desired specificity include, but are not limited to, enzyme-linkedimmunosorbent assay (ELISA) and other immunologically-mediatedtechniques known within the art. In a specific embodiment, selection ofantibodies that are specific to a particular domain of β-chemokines isfacilitated by generation of hybridomas that bind to the fragment ofβ-chemokines possessing such a domain. Antibodies that are specific forone or more domains within β-chemokines, e.g., conserved domains ofβ-chemokine family proteins, or derivatives, fragments, analogs orhomologs thereof, are also provided herein.

Anti-β-chemokine pan-antibodies may be used in methods known within theart relating to the localization and/or quantitation of a β-chemokines(e.g., for use in measuring levels of β-chemokines within appropriatephysiological samples, for use in diagnostic methods, for use in imagingthe protein, and the like). In a given embodiment, antibodies forβ-chemokines, or derivatives, fragments, analogs or homologs thereof,that contain the antibody derived binding domain, are utilized aspharmacologically-active compounds (hereinafter “Therapeutics”).

An anti-β-chemokine pan-antibody (e.g., monoclonal antibody) can be usedto isolate β-chemokines by standard techniques, such as affinitychromatography or immunoprecipitation. An anti-β-chemokine pan-antibodycan facilitate the purification of natural β-chemokines from cells andof recombinantly produced β-chemokines expressed in host cells.Moreover, as anti-β-chemokine pan-antibody can be used to detectβ-chemokines (e.g., in a cellular lysate or cell supernatant) in orderto evaluate the abundance and pattern of expression of the β-chemokines.Anti-β-chemokine pan-antibodies can be used diagnostically to monitorprotein levels in tissue as part of a clinical testing procedure inorder to, for example, determine the efficacy of a given treatmentregimen. Detection can be facilitated by coupling (i.e., physicallylinking) the antibody to a detectable substance. Examples of detectablesubstances include various enzymes, prosthetic groups, fluorescentmaterials, luminescent materials, bioluminescent materials, andradioactive materials. Examples of suitable enzymes include horseradishperoxidase, alkaline phosphatase, β-galactosidase, oracetylcholinesterase; examples of suitable prosthetic group complexesinclude streptavidin/biotin and avidin/biotin; examples of suitablefluorescent materials include umbelliferone, fluorescein, fluoresceinisothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansylchloride or phycoerythrin; an example of a luminescent material includesluminol; examples of bioluminescent materials include luciferase,luciferin, and aequorin, and examples of suitable radioactive materialinclude ¹²⁵I, ¹³¹I, ³⁵S or ³H.

In addition, the antibodies of the present invention may be conjugatedto toxins such as radioisotopes, protein toxins and chemical toxinswhich may be conjugated to antibodies. Such toxins include, but are notlimited to Lead-212, Bismuth-212, Astatine-211, Iodine-131, Scandium-47,Rhenium-186, Rhenium-188, Yttrium-90, Iodine-123, Iodine-125,Bromine-77, Indium-111, Boron-10, Actinide, ricin, adriamycin,calicheamicin, and 5-fluorouracil.

Another aspect of the present invention is directed to methods ofinducing an immune response in a mammal against a polypeptide of theinvention by administering to the mammal an amount of the polypeptidepreparation sufficient to induce an immune response. The amount will bedependent on the animal species, size of the animal, and the like butcan be determined by those skilled in the art.

Anti-Idiotypic Antibodies

Another aspect of the invention is directed to anti-idiotypic antibodiesand anti-anti-idiotypic antibodies. An anti-idiotypic antibody is anantibody that recognizes determinants of another antibody (a targetantibody). Generally, the anti-idiotypic antibody recognizesdeterminants of the antigen-binding site of the target antibody.Typically, the target antibody is a monoclonal antibody. Ananti-idiotypic antibody is generally prepared by immunizing an animal(particularly, mice) of the same species and genetic type as the sourceof the target monoclonal antibody, with the target monoclonal antibody.The immunized animal mounts an immune response to the idiotypicdeterminants of the target monoclonal antibody and produces antibodiesagainst the idiotypic determinants of the target monoclonal antibody.Antibody-producing cells, such as splenic cells, of the immunized animalmay be used to generate anti-idiotypic monoclonal antibodies.Furthermore, an anti-idiotypic antibody may also be used to immunizeanimals to produce anti-anti-idiotypic antibodies. These immunizedanimals may be used to generate anti-anti-idiotypic monoclonalantibodies using standard techniques. The anti-anti-idiotypic antibodiesmay bind to the same epitope as the original, target monoclonal antibodyused to prepare the anti-idiotypic antibody. The anti-anti-idiotypicantibodies represent other monoclonal antibodies with the same antigenspecificity as the original target monoclonal antibody.

If the binding of the anti-idiotypic antibody with the target antibodyis inhibited by the relevant antigen of the target antibody, and if theanti-idiotypic antibody induces an antibody response with the samespecificity as the target antibody, it mimics the antigen of the targetantibody. Such an anti-idiotypic antibody is an “internal imageanti-idiotypic” and is capable of inducing an antibody response as if itwere the original antigen. (Bona and Kohler, ANTI-IDIOTYPIC ANTIBODIESAND INTERNAL IMAGE, IN M ONOCLONAL AND ANTI-IDIOTYPIC ANTIBODIES: PROBES FOR RECEPTOR STRUCTURE AND FUNCTION, Venter J. C., Frasser, C. M.,Lindstrom, J. (Eds.), Alan R. Liss, N.Y., 1984. pp 141-149). Vaccinesincorporating internal image anti-idiotype antibodies have been shown toinduce protective responses against viruses, bacteria, and parasites(Kennedy et al. (1986) 232:220-223; McNamara et al. (1985) Science226:1325-1326). Internal image anti-idiotypic antibodies have also beenshown to induce immunity to tumor related antigens (Raychauhuri et al.(1986) J. Immunol. 137:1743-1749; Raychauhuri et al. (1987) J. Immunol.139:3902-3910; Bhattacharya-Chatterjee et al. (1987) J. Immunol.139:1354-1360; Bhattacharya-Chatterjee et al. (1988) J. Immunol.141:1398-1403; Herlyn, D. et al. (1989) Intern. Rev. Immunol. 4:347-357;Chen, Z.-J et al. (1990) Cell Imm. Immunother. Cancer 351-359; Herlyn,D. et al. (1991) In Vivo 5:615-624; Furuya et al. (1992) Anticancer Res.12:27-32; Mittelman A. et al. (1992) Proc. Natl. Acad. Sci., USA89:466-470; Durrant, L. G. et al. (1994) Cancer Res. 54:4837-4840;Mittelman, A. et al. (1994) Cancer Res. 54:415-421; Schmitt, H. et al.(1994) Hybridoma 13:389-396; Chakrobarty, M. et al. (1995) J.Immunother. 18:95-103; Chakrobarty, M. et al. (1995) Cancer Res.55:1525-1530; Foon, K. A. et al. (1995) Clin. Cancer Res. 1:1205-1294;Herlyn, D, et al. (1995) Hybridoma 14:159-166; Sclebusch, H. et al.(1995) Hybridoma 14:167-174; Herlyn, D. et al. (1996) Cancer ImmunolImmunother. 43:65-76).

Anti-idiotypic antibodies for β-chemokines may be prepared, for example,by immunizing an animal, such as a mouse, with a immunogenic amount of acomposition comprising β-chemokines or immunogenic portions thereof,containing at least one antigenic epitope of β-chemokines. Thecomposition may also contain a suitable adjuvant, and any carriernecessary to provide immunogenicity. Monoclonal antibodies recognizingβ-chemokines may be prepared from the cells of the immunized animal asdescribed above. A monoclonal antibody recognizing a common epitope ofβ-chemokines is then selected and used to prepare a compositioncomprising an immunogenic amount of the anti-β-chemokine monoclonalantibody. Typically, a 25 to 200 μg dose of purified β-chemokinemonoclonal would be sufficient in a suitable adjuvant.

Animals may be immunized 2-6 times at 14 to 30 day intervals betweendoses. Typically, animals are immunized by any suitable route ofadministration, such as intraperitoneal, subcutaneous, intravenous or acombination of these. Anti-idiotypic antibody production may bemonitored during the immunization period using standard immunoassaymethods. Animals with suitable titers of antibodies reactive with thetarget monoclonal antibodies may be re-immunized with the monoclonalantibody used as the immunogen three days before harvesting the antibodyproducing cells. Preferably, spleen cells are used, although otherantibody producing cells may be selected. Antibody-producing cells areharvested and fused with myeloma cells to produce hybridomas, asdescribed above, and suitable anti-idiotypic antibody-producing cellsare selected.

Anti-anti-idiotypic antibodies are produced by another round ofimmunization and hybridoma production by using the anti-idiotypicmonoclonal antibody as the immunogen.

Expression of Recombinant Antibodies

Chimeric, humanized, and human antibodies as well as antigen-bindingfragments thereof, are typically produced by recombinant expression.Nucleic acids encoding humanized light and heavy chain variable regions,optionally linked to constant regions, are inserted into expressionvectors. The light and heavy chains can be cloned in the same ordifferent expression vectors. The DNA segments encoding immunoglobulinchains are operably linked to control sequences in the expressionvector(s) that ensure the expression of immunoglobulin polypeptides.Expression control sequences include, but are not limited to, promoters(e.g., naturally-associated or heterologous promoters), signalsequences, enhancer elements, and transcription termination sequences.Preferably, the expression control sequences are eukaryotic promotersystems in vectors capable of transforming or transfecting eukaryotichost cells. Once the vector has been incorporated into the appropriatehost, the host is maintained under conditions suitable for high levelexpression of the nucleotide sequences, and the collection andpurification of the crossreacting antibodies.

These expression vectors are typically replicable in the host organismseither as episomes or as an integral part of the host chromosomal DNA.Commonly, expression vectors contain selection markers (e.g.,ampicillin-resistance, hygromycin-resistance, tetracycline resistance orneomycin resistance) to permit detection of those cells transformed withthe desired DNA sequences (see, e.g., Itakura et al., U.S. Pat. No.4,704,362).

E. coli is one prokaryotic host particularly useful for cloning thepolynucleotides (e.g., DNA sequences) of the present invention. Othermicrobial hosts suitable for use include bacilli, such as Bacillussubtilus, and other enterobacteriaceae, such as Salmonella, Serratia,and various Pseudomonas species. In these prokaryotic hosts, one canalso make expression vectors, which will typically contain expressioncontrol sequences compatible with the host cell (e.g., an origin ofreplication). In addition, any number of a variety of well-knownpromoters will be present, such as the lactose promoter system, atryptophan (trp) promoter system, a beta-lactamase promoter system, or apromoter system from phage lambda. The promoters will typically controlexpression, optionally with an operator sequence, and have ribosomebinding site sequences and the like, for initiating and completingtranscription and translation.

Other microbes, such as yeast, are also useful for expression.Saccharomyces is a preferred yeast host, with suitable vectors havingexpression control sequences (e.g., promoters), an origin ofreplication, termination sequences and the like as desired. Typicalpromoters include 3-phosphoglycerate kinase and other glycolyticenzymes. Inducible yeast promoters include, among others, promoters fromalcohol dehydrogenase, isocytochrome C, and enzymes responsible formaltose and galactose utilization.

In addition to microorganisms, mammalian tissue cell culture may also beused to express and produce the polypeptides of the present invention(e.g., polynucleotides encoding immunoglobulins or fragments thereof).See Winnacker, From Genes to Clones, VCH Publishers, N.Y., N.Y. (1987).Eukaryotic cells are actually preferred, because a number of suitablehost cell lines capable of secreting heterologous proteins (e.g., intactimmunoglobulins) have been developed in the art, and include CHO celllines, various Cos cell lines, HeLa cells, preferably, myeloma celllines, or transformed B-cells or hybridomas. Preferably, the cells arenonhuman. Expression vectors for these cells can include expressioncontrol sequences, such as an origin of replication, a promoter, and anenhancer (Queen et al., Immunol. Rev. 89:49 (1986)), and necessaryprocessing information sites, such as ribosome binding sites, RNA splicesites, polyadenylation sites, and transcriptional terminator sequences.Preferred expression control sequences are promoters derived fromimmunoglobulin genes, SV40, adenovirus, bovine papilloma virus,cytomegalovirus and the like. See Co et al., J. Immunol. 148:1149(1992).

Alternatively, antibody-coding sequences can be incorporated intransgenes for introduction into the genome of a transgenic animal andsubsequent expression in the milk of the transgenic animal (see, e.g.,Deboer et al., U.S. Pat. No. 5,741,957, Rosen, U.S. Pat. No. 5,304,489,and Meade et al., U.S. Pat. No. 5,849,992). Suitable transgenes includecoding sequences for light and/or heavy chains in operable linkage witha promoter and enhancer from a mammary gland specific gene, such ascasein or beta lactoglobulin.

The vectors containing the polynucleotide sequences of interest (e.g.,the heavy and light chain encoding sequences and expression controlsequences) can be transferred into the host cell by well-known methods,which vary depending on the type of cellular host. For example, calciumchloride transfection is commonly utilized for prokaryotic cells,whereas calcium phosphate treatment, electroporation, lipofection,biolistics or viral-based transfection may be used for other cellularhosts. (See generally Sambrook et al., Molecular Cloning: A LaboratoryManual (Cold Spring Harbor Press, 2nd ed., 1989) (incorporated byreference is its entirety for all purposes). Other methods used totransform mammalian cells include the use of polybrene, protoplastfusion, liposomes, electroporation, and microinjection (see generally,Sambrook et al., supra). For production of transgenic animals,transgenes can be microinjected into fertilized oocytes, or can beincorporated into the genome of embryonic stem cells, and the nuclei ofsuch cells transferred into enucleated oocytes.

When heavy and light chains are cloned on separate expression vectors,the vectors are co-transfected to obtain expression and assembly ofintact immunoglobulins. Once expressed, the whole antibodies, theirdimers, individual light and heavy chains, or other immunoglobulin formsof the present invention can be purified according to standardprocedures of the art, including ammonium sulfate precipitation,affinity columns, column chromatography, HPLC purification, gelelectrophoresis and the like (see generally Scopes, Protein Purification(Springer-Verlag, N.Y., (1982)). Substantially pure immunoglobulins ofat least about 90 to 95% homogeneity are preferred, and 98 to 99% ormore homogeneity most preferred, for pharmaceutical uses.

Chemical Modifications

In some embodiments, the antibodies and antibody fragments of theinvention may be chemically modified to provide a desired effect. Forexample, pegylation of antibodies and antibody fragments of theinvention may be carried out by any of the pegylation reactions known inthe art, as described, for example, in the following references: Focuson Growth Factors 3:4-10 (1992); EP 0 154 316; and EP 0 401 384 (each ofwhich is incorporated by reference herein in its entirety). Preferably,the pegylation is carried out via an acylation reaction or an alkylationreaction with a reactive polyethylene glycol molecule (or an analogousreactive water-soluble polymer). A preferred water-soluble polymer forpegylation of the antibodies and antibody fragment of the invention ispolyethylene glycol (PEG). As used herein, “polyethylene glycol” ismeant to encompass any of the forms of PEG that have been used toderivatize other proteins, such as mono (Cl—ClO) alkoxy- oraryloxy-polyethylene glycol.

Methods for preparing pegylated antibodies and antibody fragments of theinvention will generally comprise the steps of (a) reacting the antibodyor antibody fragment with polyethylene glycol, such as a reactive esteror aldehyde derivative of PEG, under conditions whereby the antibody orantibody fragment becomes attached to one or more PEG groups, and (b)obtaining the reaction products. It will be apparent to one of ordinaryskill in the art to select the optimal reaction conditions or theacylation reactions based on known parameters and the desired result.

Pegylated antibodies and antibody fragments may generally be used totreat conditions that may be alleviated or modulated by administrationof the antibodies and antibody fragments described herein. Generally thepegylated antibodies and antibody fragments have increased half-life, ascompared to the nonpegylated antibodies and antibody fragments. Thepegylated antibodies and antibody fragments may be employed alone,together, or in combination with other pharmaceutical compositions.

In other embodiments of the invention the antibodies or antigen-bindingfragments thereof are conjugated to albumen using art recognizedtechniques.

In another embodiment of the invention, antibodies, or fragments,thereof, are modified to reduce or eliminate potential glycosylationsites. Such modified antibodies are often referred to as “aglycosylated”antibodies. In order to improve the binding affinity of an antibody orantigen-binding fragment thereof, glycosylation sites of the antibodycan be altered, for example, by mutagenesis (e.g., site-directedmutagenesis). “Glycosylation sites” refer to amino acid residues whichare recognized by a eukaryotic cell as locations for the attachment ofsugar residues. The amino acids where carbohydrate, such asoligosaccharide, is attached are typically asparagine (N-linkage),serine (O-linkage), and threonine (O-linkage) residues. In order toidentify potential glycosylation sites within an antibody orantigen-binding fragment, the sequence of the antibody is examined, forexample, by using publicly available databases such as the websiteprovided by the Center for Biological Sequence Analysis (seehttp://www.cbs.dtu.dk/services/NetNGlyc/ for predicting N-linkedglycoslyation sites) and http://www.cbs.dtu.dk/services/NetOGlyc/ forpredicting O-linked glycoslyation sites). Additional methods foraltering glycosylation sites of antibodies are described in U.S. Pat.Nos. 6,350,861 and 5,714,350.

In yet another embodiment of the invention, antibodies or fragmentsthereof can be altered wherein the constant region of the antibody ismodified to reduce at least one constant region-mediated biologicaleffector function relative to an unmodified antibody. To modify anantibody of the invention such that it exhibits reduced binding to theFc receptor, the immunoglobulin constant region segment of the antibodycan be mutated at particular regions necessary for Fc receptor (FcR)interactions (see e.g., Canfield, S. M. and S. L. Morrison (1991) J.Exp. Med. 173:1483-1491; and Lund, J. et al. (1991) J. of Immunol.147:2657-2662). Reduction in FcR binding ability of the antibody mayalso reduce other effector functions which rely on FcR interactions,such as opsonization and phagocytosis and antigen-dependent cellularcytotoxicity.

Therapeutics

Antibodies or antigen-binding fragments of the invention are useful for,e.g., therapeutic purposes (by modulating activity of monocytechemotactic proteins), diagnostic purposes to detect or quantifymonocyte chemotactic proteins, and purification of monocyte chemotacticproteins. Therefore, kits comprising an antibody of the invention forany of the purposes described herein are also within the scope of theinvention.

The β-chemokines, particularly MCP-1, MCP-2 and MCP-3 have been shown toplay a role in pathological conditions associated with inflammation (VanCoillie et al. (1999) Cytokine & Growth Factor Rev. 10:61-86). MCP-1,MCP-2, and MCP-3 have all been shown to have potent chemotactic activityfor leukocytes, especially monocytes (van Coillie et al. (1999) Cytokine& Growth Factor Rev. 10:61-86). All three chemokines also share otherfunctions (e.g., glucosaminidase release, gelatinase B release, granzymeA) which combined with their chemotactic activity enable leukocytes tomigrate into tissues and towards site of inflammation. Recruitment ofleukocytes to inflammatory sites is thought to contribute greatly to theinflammatory process. Inhibition of leukocyte recruitment via MCP-1antagonism (e.g., in MCP-1 knockout animals and in MCP-1 depletionstudies using anti-MCP-1 mAbs) has been shown to reduce leukocyteinfiltration (particularly monocyte recruitment) and is correlated withreduction in disease (van Coillie et al. (1999) Cytokine & Growth FactorRev. 10:61-86).

Like MCP-1, MCP-2 and MCP-3 are also molecules with potent chemotacticactivity for monocytes, T lymphocytes, and basophils. Given theiroverlapping activities and the increased expression of all threechemokines (MCP-1, MCP-2, and MCP-3) in human disease, blockade of allthree MCP molecules is expected to have a greater beneficial effect thanjust inhibition of MCP-1 alone. Blockade of multiple MCP molecules(MCP-1, MCP-2 and MCP-3) should more efficiently inhibit recruitment ofcertain cell types for which MCP-1 is a poor chemotactic stimulus. Thus,while MCP-1 does not efficiently Induce migration of eosinophils orresting neutrophils, MCP-2 is a potent chemotactic stimulus foreosinophils, and MCP-3 shows activity against both eosinophils andneutrophils (van Coillie et al. (1999) Cytokine & Growth Factor Rev.10:61-86).

Accordingly, the antibodies and antibody fragments of the invention areuseful to modulate the activity of these chemokines and affect thepathology of disorders associated with these chemokines. As such, theseantibodies and fragments are useful, in therapeutic compositions for thetreatment of inflammatory conditions and pathological conditionsassociated with expression of MCP molecules. In these embodiments, apatient is identified as having one of the diseases to be treated, suchas by exhibiting at least one sign or symptom of the disease ordisorder. At least one antibody or antigen-binding fragment thereof ofthe invention or compositions comprising at least one antibody orantigen-binding fragment thereof of the invention is administered in asufficient amount to alleviate at least one symptom of the disease ordisorder, or to reduce the activity of at least one of MCP-1, MCP-2 orMCP-3.

Disorders Amenable to Prevention or Treatment

As used herein, the terms “a disorder in which MCP activity isdetrimental” and “an MCP-associated disorder” are intended to includediseases and other disorders in which the presence of MCP, includingMCP-1, MCP-2, and/or MCP-3, in a subject suffering from the disorder hasbeen shown to be or is suspected of being either responsible for thepathophysiology of the disorder or a factor that contributes thedisorder. Accordingly, a disorder in which MCP activity is detrimentalis a disorder is which inhibition of MCP activity is expected to preventor alleviate the symptoms and/or progression of the disorder. Suchdisorders may be evidenced, for example, by an increase in theconcentration of MCP in a biological fluid of a subject suffering fromthe disorder (e.g., an increase in the concentration of MCP-1 in serum,plasma, synovial fluid, urine, etc. of the subject), which can bedetected, for example, using an anti-MCP antibody as described above.There are numerous examples of disorders in which MCP activity isdetrimental. The use of the antibodies and antibody portions of theinvention in the prevention or treatment of specific disorders isdiscussed further below.

Fibrotic Disease

In one embodiment of the invention, an antibody or antigen-bindingfragment thereof, having binding specificity for MCP-1, MCP-2 and/orMCP-3, an antibody or antigen-binding fragment comprising CDRs fromeither the 1A1 or 11K2 antibodies, is used in a method of prevention ortreatment of a patient suffering from a fibrotic disease. A “fibroticdisease” as used herein includes any condition marked by an increase ofinterstitial fibrous tissue. MCPs are known to be associated withfibrotic conditions. For example, MCP-1 is a potent chemoattractant formonocytes and has been implicated in a variety of inflammatory andfibrotic diseases, the pathogenesis of which is known to involveinfiltration and activation of monocytes (Zhang, et al (1994) J.Immunol. 153:4733-4741). Along with increased TGF-β and collagenproduction, fibrotic diseases are also characterized by increased levelsof MCP-1 (Antoniades et al. (1992) J. Immunol 89:5371-5375; Wada et al.(1996) FASEB J. 10:1418-1425; Saitoh et al. (1998) J. Clin. Lab. Anal.12:1-5; Hasegawa et al. (1999) Clin. Exp. Immunol. 117:159-165; Wada etal. (1999) Kidney Int. 65:995-1003; Wada et al. (2000) Kidney Int.58-1492-4499). Increased expression of MCP-1 during fibrotic diseaseshas been well characterized in both human and in rodent models. Inhumans, MCP-1 is up-regulated in idiopathic pulmonary fibrosis(Antoniades et al., supra), IgA nephropathy (Saitoh et al., supra),diabetic nephropathy (Wada et al. (2000), supra), lupus nephritis (Wadaet al. (1996), supra), crescentic glomerulonephritis (Wada, 1999),supra), and scleroderma (Hasegawa, supra). While not expressed in normaltissues, MCP-1 was highly expressed in the fibrotic skin and lungs ofscleroderma patients, and the elevated levels of MCP-1 found in patientserum correlated with the presence of fibrosis and with earlier onset ofscleroderma (Hasegawa, supra). MCP-1 expression also correlatedpositively with severity of renal fibrosis in diseases such as IgAnephropathy, diabetic nephropathy, lupus nephritis, and crescenticglomerulonephritis.

Oncogenic Disease

In another embodiment of the invention, an antibody or antigen-bindingfragment thereof, having binding specificity for MCP-1, MCP-2 and/orMCP-3, e.g., an antibody or antigen-binding fragment comprising CDRsfrom either the 1A1 or 11K2 antibodies, is used in a method ofprevention or treatment of a patient suffering from an oncogenic diseaseor cancer. MCPs are known to be associated with oncogenic conditions.For example, MCP-1 is a potent inducer of angiogenesis and plays animportant role in tumor growth. Evidence for a role of MCP-1 intumorigenesis involved treatment of immunodeficient mice bearing MCP-1producing human breast carcinoma cells with neutralizing anti-MCP-1 mAb(Salcedo, (2000) Blood 96:34-40). Treatment with anti-MCP-1 mAb resultedin significant increases in animal survival (mean survival increasedfrom 45 days to 75 days) and marked inhibition of tumor metastasis (60%decrease in lung metastatic index).

Immunopathologic Disease

In another embodiment of the invention, an antibody or antigen-bindingfragment thereof, having binding specificity for MCP-1, MCP-2 and/orMCP-3, e.g., an antibody or antigen-binding fragment comprising CDRsfrom either the 1A1 or 11K2 antibodies, is used in a method ofprevention or treatment of a patient suffering from an immunopathologicdisease. An “immunopathologic disease” as used herein is defined as anycondition associated with an immune response which is related to adisease. MCPs have been associated with immunopathologic conditions. Forexample, there is a strong link between MCP-1 expression andimmunopathologic disease in humans. Experiments usinggenetically-engineered mice and in vivo data using function-blockingantibodies to MCP-1 provide evidence supporting the validity of MCP-1antagonism in a variety of diseases characterized by mononuclearinfiltration. Included among these diseases is: atherosclerosis (MCP-1KO, CCR2 KO), arthritis (MCP-1 mAb), asthma (MCP-1 mAb),glomerulonephritis (MCP-1 KO, MCP-1 mAb), lupus nephritis (MCP-1 KO) andmultiple sclerosis (MCP-1 KO, MCP-1 mAb, CCR2 KO) (see, for example, Luet al. (1998) J. Exp. Med. 187601-608); Kurihara et al. (1997) J. Exp.Med. 186:1757-1762; Boring et al. (1997) J. Clin. Invest.100:2552-2561); Kuziel et al. (1997) PNAS 94:12053-12058; Blease et al.(2000) J. Immunol. 165:2603-2611; Traynor et al. (2000) J. Immunol.164:2021-2027; Boring et al. (1998) Nature 394:894-897; Dawson et al.(1999) Atherosclerosis 143:205-211; Fife et al. (2000) J. Exp. Med. 192:899-905; Izikson et al. (2000) J. Exp. Med. 192:1075-1080; Bird et al.(2000) Kidney Int. 57:129-136; MacLean et al. (2000) J. Immunol.165:165:6568-6575; Karpus et al. (1997) J. Leukoc. Biol. 62:681-687;Gonzalo et al. (1998) J. Exp. Med. 188:157-167). In all these cases,interference with the MCP-1 pathway resulted in dramatically reducedleukocyte infiltration, with monocyte recruitment being particularlyaffected. This dramatic reduction in monocyte recruitment correlatedwell with reduction in disease.

Other Disorders

In certain embodiments, the antibodies or antigen-binding fragments ofthe present invention are useful in the prevention or treatment ofglomerulonephritis, scleroderma, cirrhosis, multiple sclerosis, lupusnephritis, atherosclerosis, inflammatory bowel diseases or rheumatoidarthritis. In another embodiment, the antibodies or antigen-bindingfragments of the invention can be used to treat or prevent inflammatorydisorders, including, but not limited to, Alzheimer's, severe asthma,atopic dermatitis, cachexia, CHF-ischemia, coronary restinosis, Crohn'sdisease, diabetic nephropathy, lymphoma, psoriasis,fibrosis/radiation-induced, juvenile arthritis, stroke, inflammation ofthe brain or central nervous system caused by trauma, and ulcerativecolitis. Other inflammatory disorders which can be prevented or treatedwith the antibodies or antigen-binding fragments of the inventioninclude inflammation due to corneal transplantation, chronic obstructivepulmonary, disease, hepatitis C, multiple myeloma, and osteoarthritis.In another embodiment, the antibodies or antigen-binding fragments ofthe invention can be used to prevent or treat neoplasia, including, butnot limited to bladder cancer, breast cancer, head and neck cancer,kaposi's sarcoma, melanoma, ovarian cancer, small cell lung cancer,stomach cancer, leukemia/lymphoma, and multiple myeloma. Additionalneoplasia conditions include, cervical cancer, colo-rectal cancer,endometrial cancer, kidney cancer, non-squamous cell lung cancer, andprostate cancer. In another embodiment, the antibodies orantigen-binding fragments of the invention can be used to prevent ortreat fibrotic disorders, including, but not limited to CHF-ischemia,coronary restenosis, diabetic vasculopathy, myocardialinfarction/unstable angina, and radiation fibrosis. Additional examplesof fibrotic disorders which can be treated in accordance with theinvention include diabetic nephropathy, and impotence (Peyronie's). Inanother embodiment, the antibodies or antigen-binding fragments of theinvention can be used to prevent or treat neurodegenerative disorders,including, but not limited to Alzheimer's, stroke, and traumatic brainor central nervous system injuries. Additional neurodegenerativedisorders include ALS/motor neuron disease, diabetic peripheralneuropathy, diabetic retinopathy, Huntington's disease, maculardegeneration, and Parkinson's disease.

In clinical applications, a patient is identified as having or at riskof developing a disease or disorder associated with detrimental MCPactivity, such as by exhibiting at least one sign or symptom of thedisease or disorder. At least one antibody or antigen-binding fragmentthereof of the invention or compositions comprising at least oneantibody or antigen-binding fragment thereof of the invention isadministered in a sufficient amount to treat at least one symptom of thedisease or disorder, or to reduce the activity of at least one of MCP-1,MCP-2 or MCP-3.

Moreover, an antibody of the invention can be administered to anon-human mammal expressing a chemokine with which the antibodycross-reacts (e.g., a primate, pig or mouse) for veterinary purposes oras an animal model of human disease. Regarding the latter, such animalmodels may be useful for evaluating the therapeutic efficacy ofantibodies of the invention (e.g., testing of dosages and time coursesof administration). Examples of animal models which can be used forevaluating the therapeutic efficacy of antibodies or antigen-bindingfragments of the invention for preventing or treating glomerulonephritisinclude anti-GBM-induced glomerulonephritis (Wada et al. (1996) KidneyInt. 49:761-767) and anti-thyl-induced glomerulonephritis (Schneider etal. (1999) Kidney Int. 56:135-144). Examples of animal models which canbe used for evaluating the therapeutic efficacy of antibodies orantigen-binding fragments of the invention for preventing or treatingcirrhosis include carbon tetrachloride-induced cirrhosis and liverfibrosis (Sakadamis et al. (2001) Res Exp Med 200:137-54). Examples ofanimal models which can be used for evaluating the therapeutic efficacyof antibodies or antigen-binding fragments of the invention forpreventing or treating multiple sclerosis include experimentalautoimmune encephalomyelitis (EAE) (Link and Xiao (2001) Immunol. Rev.184:117-128). Animal models can also be used for evaluating thetherapeutic efficacy of antibodies or antigen-binding fragments of theinvention for preventing or treating lupus, for example using theMRL-Fas^(Ipr) mice (Schneider, supra; Tesch et al. (1999) J. Exp. Med.190). Examples of animal models which can be used for evaluating thetherapeutic efficacy of antibodies or antigen-binding fragments of theinvention for preventing or treating atherosclerosis include using micedeficient in apolipoprotein A, ApoE, and LDL R_(L) (Dansky et al. (1999)Arterioscler Thromb. Vasc. Biol. 19:1960-1968; Lou et al. (1998) PNAS95:12591-12595). Examples of animal models which can be used forevaluating the therapeutic efficacy of antibodies or antigen-bindingfragments of the invention for preventing or treating inflammatory boweldisease (IBD) include TNBS-induced IBD, DSS-induced IBD, and (Padol etal. (2000) Eur. J. Gastrolenterol. Hepatol. 12:257; Murthy et al. (1993)Dig. Dis. Sci. 38:1722). Examples of animal models which can be used forevaluating the therapeutic efficacy of antibodies or antigen-bindingfragments of the invention for preventing or treating rheumatoidarthritis (RA) include adjuvant-induced RA, collagen-induced RA, amdcollagen mAb-induced RA (Holmdahl et al., (2001) Immunol. Rev. 184:184;Holmdahl et al., (2002) Ageing Res. Rev. 1:135; Van den Berg (2002)Curr. Rheumatol. Rep. 4:232).

In addition, animal models for evaluating the efficacy of antibodies orantigen-binding fragments of the invention in treating or preventinghuman fibrotic diseases, include rodent models of pulmonary (Brieland etal. (1992) Am. J. Respir. Cell. Mol. Biol. 7:134-139; Zhang et al.(1994) J. Immunol. 153:4733-4741; Johnston et al. (1998) Exp. Lung Res.24:321-337), vascular (Furukawa et al. (1999) Circ. Res. 84:306-314),and renal (Lloyd et al. (1997) J. Exp. Med. 185:1371-1380; Fujinaka etal. (1997) J. Am. Soc. Nephrol. 8:1174-1778; Schneider, supra; Tesch etal. (1999) J. Exp. Med. 190:1813-1824; Tesch et al. (1999) J. Clin.Invest. 103:73-80) fibrosis. Alport's model of renal fibrosis can alsobe used to evaluate the efficacy of the antibodies or antigen-bindingfragments of the invention.

The therapeutic compositions of the invention include at least oneantibody or antibody fragment of the invention in a pharmaceuticallyacceptable carrier. A “pharmaceutically acceptable carrier” refers to atleast one component of a pharmaceutical preparation that is normallyused for administration of active ingredients. As such, a carrier maycontain any pharmaceutical excipient used in the art and any form ofvehicle for administration. The compositions may be, for example,injectable solutions, aqueous suspensions or solutions, non-aqueoussuspensions or solutions, solid and liquid oral formulations, salves,gels, ointments, intradermal patches, creams, lotions, tablets,capsules, sustained release formulations, and the like. Additionalexcipients may include, for example, colorants, taste-masking agents,solubility aids, suspension agents, compressing agents, entericcoatings, sustained release aids, and the like.

The therapeutic compositions of the invention may be administered by anyacceptable route of administration including, but not limited tointravenous, intradermal, interperitoneal, enteric, vaginal, rectal,nasal, transdermal, subcutaneous, and intramuscular.

Methods for Screening of β-Chemokine Pan-Antibodies

The pan-antibodies and fragments thereof of the invention may be assayedfor specific binding to the β-chemokines, particularly MCPs (e.g.,MCP-1, MCP-2 and MCP-3) in competitive and non-competitive bindingimmunoassays. Well-known procedures for immunoassays may be found, forexample, in Stites and Terr (Eds.) BASIC AND CLINICAL IMMUNOLOGY (7thed.), 1991; Maggio (Ed.) ENZYME IMMUNOASSAY, CRC Press, Boca Raton,Fla., 1980; Tijan (1985) PRACTICE AND THEORY OF ENZYME IMMUNOASSAYS,LABORATORY TECHNIQUES IN BIOCHEMISTRY AND MOLECULAR BIOLOGY, ElsevierScience Publishers B.V., Amsterdam; Harlow and Lane (Eds.) ANTIBODIES, ALABORATORY MANUAL, Cold Spring Harbor, N.Y., 1988; Chan (Ed.) (1987)IMMUNOASSAY: A PRACTICAL GUIDE, Academic Press, Orlando, Fla., 1987;Price and Newman (Eds.) PRINCIPLES AND PRACTICE OF IMMUNOASSAYS,Stockton Press, N.Y. 1991; and Ngo (Ed.) (1988) NON-ISOTOPICIMMUNOASSAYS, Plenum Press, N.Y., 1988; the disclosures of which areincorporated by reference herein.

Immunoassays to measure antibody binding can be either competitive ornoncompetitive. In general in the antibody context, a competitive assayinvolves competition for binding of a ligand between two antibodies. Forexample, a labeled MCP-1 may be used to assess whether one antibody cancompete with another antibody for binding the labeled MCP-1. The assaymay be based on such standard assays as the enzyme linked immunosorbentassay (ELISA) or radioimmunoassay (RIA) for example.

Alternatively, the antibodies and antibody fragments of the inventionmay be tested in noncompetitive assays for binding to substrates. Forinstance, a standard ELISA may be used in which the ligand (e.g., MCP-1)is immobilized on an ELISA plate. A test antibody is incubated with theligand and allowed to bind. The plate is washed and thereafter, anenzyme-conjugated, secondary antibody (e.g., a mouse anti-human Fcantibody) binds to the test antibody if the test antibody is bound tothe ligand. After washing, a substrate for the enzyme is added andallowed to react with the enzyme. Generally a color change indicates thepresence of an antibody that reacts with the ligand. The ELISA may berepeated for different β-chemokines to determine which chemokines arerecognized by the test antibody.

Immunoassays often use labeled assay components. The label can be in avariety of forms and may be coupled directly or indirectly to thedesired component of the assay according to methods well known in theart. Common labels for assay components include radioactive isotopes,including ³H, ¹²⁵I, ³⁵S, ¹⁴C, and ³²P, fluorophores, chemiluminescentagents, and enzymes. The choice of a particular label will depend on thesensitivity required, the case of conjugation with the compound, thestability requirements, and the available instrumentation, and will beeasily determined by one of ordinary skill in the art.

In order to determine that the monoclonal antibodies do not bind theMRHAS motifs, an ELISA for MRHAS family members, such as regions ofrubella structural proteins, HIV gag, HIV envelope, Haemophilusinfluenzae lipoprotein Neisseria menigitidis POPM3, Streptococcuspneumoniae Protein A, and Listeria monocytogenes protein precursor, asshown in WO 95/09232. Those antibodies that bind to a non-chemokineMHRAS member are not antibodies of the invention.

An example of a screening assay is set forth in the Examples, but theinvention is not limited thereto or thereby.

Assays to assess whether the antibodies of the invention inhibitβ-chemokine activity, particularly MCP activity (e.g., MCP-1, MCP-2 andMCP-3 activity) may be easily performed using known assays forchemotaxis, intracellular calcium increase, and the like.

For example, but not by way of limitation, chemokine chemotaxis assaysmay be performed in 48 well plastic chambers. The wells are separated bya filter into two compartments. The filter allows the passage of cellsfrom one compartment to the next in response to chemical gradients. Testcells are placed in one compartment of the chamber in a culture mediumand an MCP, for example, is placed in culture medium in the othercompartment. Cells adhering to the filter or traversing the filter arecounted. In other wells, the MCP is mixed with the test antibody todetermine if the antibody is able to block cell migration.

In vitro Assays

The antibodies and antibody fragments of the invention may be used todetect MCPs in samples using a variety of well-known immunologicalassays. The antibodies may be used, for example, in ELISAs, Westernblots, radioimmunoassays, immunoprecipitaton, immunoaffinitychromatography, immunostaining of tissue sections, immunogold detectionin tissue samples with electron microscopy, and the like. The protocolsfor these and other assays are well-known in the art and are well withinthe purview of the skilled artisan.

The immunoassays using the antibodies and antibody fragments of theinvention may be used to detect the presence and relative amounts ofβ-chemokines in a sample. Samples may include, but are not limited to,homogenized tissue or cells, histological tissue sections for light andelectron microscopy, protein extracts of tissue or cells, serum, blood,and the like. The presence of increased amounts of a β-chemokine(s)relative to normal samples, for example, may indicate the presence of adisease state, and treatment with a therapeutic of the invention may beindicated. In some instances, there may be a decreased amount ofβ-chemokine(s) relative to normal samples, and treatment withappropriate β-chemokine(s) or internal image antibodies that mimicβ-chemokine(s) may be used to stimulate immune function.

In some embodiments, an immunoassay may be used to aid in thepurification of β-chemokines. For example, an immunoaffinity resin maybe used in which the antibodies or antibody fragments of the inventionare immobilized on a substrate. A sample containing the β-chemokine(s)is added to the immunoaffinity resin and the antibodies become bound tothe resin, while other components of the sample remain in solution. Theresin is washed and the β-chemokines are subsequently eluted from theresin, substantially purified and isolated. Preferably, the antibodiesused in the immunoassay will have high binding affinity, as definedherein.

This invention is further illustrated by the following examples whichshould not be construed as limiting. The contents of all references,patents and published patent applications cited throughout thisapplication, as well as the figures and the Sequence Listing, areincorporated herein by reference.

EXAMPLES

I. Characterization of Anti-Chemokine Monoclonal Antibody Supernatants

Example 1 ELISA Screening

MaxiSorp 384 well plates were coated with 15-20 μl antigen in PBS.Recombinant purified human antigens included: MCP-1, MCP-2, MCP-3,MCP-4, IL-8, eotaxin, fractalkine, Gcp-2, DC-CK (Gcp-2 and DC-CK arealso chemokines). Plates were incubated with antigen for 2 hours at 37°C. or overnight at 4° C. Non-specific sites were blocked with 80 μl/wellof 1% BSA/PBS for 1 hour at room temperature. Plates were washed and 15μl of hybridoma supernatant was added to each well and incubated for 1hour at room temperature. Plates were washed and wells were incubatedwith 20 μl/well of a 1:25 000 dilution of goat anti-mouse IgG peroxidaseconjugate (Jackson Catalog Number 515-036-003). Plates were incubatedfor 1 hour at room temperature, washed, and 20 μl/well of substrate(TMB, tetramethylbenzidine, Jackson, Catalog Number 515-036-062) wasadded. Reaction was allowed to proceed and stopped by addition of 20μl/well of 2M H₂SO₄. Reactive clones were picked for further analysis.Isotyping of hybridoma supernatants was performed by antigen-dependentELISA. Briefly, wells were coated with 50 μl of human MCP-1 (5 μg/ml)for 1 hour at 37° C. Wells were washed 4 times and blocked withPBS/1%BSA. Isotyping of hybridoma supernatants was then performed usinga mouse immunoglobulin screening/isotyping kit (Zymed Laboratories, SanFrancisco, Calif.) as recommended by the manufacturer. Specificities ofthe antibodies and clones obtained are shown in Table 1.

TABLE 1 Panel of MCP mAbs Specificity Block MCP-4/IL-8 MCP-1Eotaxin/Frac Affinity ligand Fusion Immunogen Clone MCP-1 MCP-2 MCP-3Gcp-2/DC-CK (Biacore) Sub-Type binding IA1 MCP-1 1M-11 ++++ − − − ++++IgG1 ++++ 3N10 ++++ − − − ++++ IgG1 ++++ IA7 MCP-1 11K2 ++++ ++++ ++++ −++++ IgG1 ++++ MCP-2 7F7 ++++ − − − ++++ IgG1 +++ MCP-3 6D21 ++++ ++++++ − ++++ IgG1 ++++ 6B11 ++ − + − +++ IgG1 +++ 1A1 +++ + + − ++++ IgG1++++ IA8 MCP-1 4N4 ++++ ++ ++ − ++++ IgG1 ++++ MCP-2 5A13 +++ ++ ++ −+++ IgG1 ++++ IA9 MCP-1 5J23 ++++ ++ − − ++++ IgG1 ++++ MCP-2 6I5 +++++++ +++ − ++++ IgG1 ++++ MCP-3 7H1 ++++ ++++ ++++ − ++++ IgG1 ++++ IA10MCP-1 4N9 + − ++ − ++++ IgG1 +++ MCP-2 2O24 ++ − ++ − ++++ IgG1 +++MCP-3 9H23 ++ − + − +++ IgG1 +++ 9B11 ++++ − ++++ − ++++ IgG1 +++ 9B12 +− +++ − ++++ IgG1 +++ 9C11 ++++ − ++++ − ++++ IgG1 +++ 10D18 ++++ − ++++− ++++ IgG1 ++++ 12F15 +++ − +++ − ++++ IgG1 ++++ MCP-1 D9 ++++ − − −(MCP−4, IL−8 +++ and cotaxin only tested) + and − indicate the relativeamount of binding of the antibody to the various immobilized ligands.

In addition, both 1A1 and 11K2 mAbs recognize primate MCP-1. Plate werecoated with 1 μg/ml of chemically synthesized chemokines (correspondingto the cynomolgus and rhesus MCP-1 sequences) and probed with 10 μg/mlof monoclonal antibodies, including MOPC21, 11K2, 3N10, 1A1, D9, and1M11, as described above. Results demonstrate that all of the abovemAbs, including 1A1 and 11K2) and with the exception of the isotypecontrol mAb MOPC21, also recognize primate MCP-1.

Example 2 Binding Assay

¹²⁵I labeled MCP-1 (2200 Ci/mmol) was purchased from NEN Life Sciences(Boston, Mass.). Hybridoma supernatants (50 μl) were pre-incubated with1 nM ¹²⁵I MCP-1 (50 μl) for 60 minutes at room temperature prior to theaddition of the CCR2-expressing human monocyte cell line, THP-1. THP-1cells (1×10⁷ cells/ml; 50 μl) were resuspended in binding buffer (50 mMHepes, 1 mM CaCl₂, 5 mM MgCl₂, 0.5% BSA) and added to the combination of¹²⁵I labeled MCP-1 and hybridoma supernatant, and incubated at 4° C. for60 minutes. Cells were then washed 3 times by centrifugation in washbuffer (50 mM Hepes, 1 mM CaCl₂, 5 mM MgCl₂, 500 mM NaCl and 0.5% BSA).Amount of bound ¹²⁵I labeled MCP-1 was then quantitated for γ-emission.Pre-incubation of THP-1 cells (1×10⁷ cells/ml; 50 μl) with unlabeledMCP-1 (500 nM; 50 μl) for 60 minutes at 4° C. prior to addition of ¹²⁵Ilabeled MCP-1 (1 nM; 50 μl) served as a negative control. The positivecontrol represents binding of ¹²⁵I labeled MCP-1 to THP-1 cells in theabsence of MCP-1 hybridoma supernatant. The results are shown in Table2.

TABLE 2 Data of block ligand ([I¹²⁵] MCP-1) binding assay from γ CounterCPM CPM Fusion Immunogen Clone # (30/10/00) (22/11/00) IA1 MCP-1 1M-11499 456 45 40 3N10 379 495 50 46 IA7 MCP-1 11K2 103 148 50 49 MCP-2 7F7394 199 189 197 MCP-3 6D21 145 108 47 42 6E11 850 894 378 323 1A1 194772 47 52 IA8 MCP-1 4N4 40 47 MCP-2 5A13 50 44 IA9 MCP-1 5J23 478 280 5947 MCP-2 6I5 677 678 44 31 MCP-3 7H1 53 207 59 77 IA10 MCP-1 4N9 776 987306 340 MCP-2 2O24 936 869 226 357 MCP-3 9H23 293 226 9B11 679 892 238201 9B12 834 657 304 265 9C11 605 512 241 252 10D18 485 444 174 31012F15 421 344 281 213 12K14 406 918 Negative control 836 461 68 143Positive control 5861 3447 2084 2933

Example 3 Inhibition of Chemotaxis in Response to MCP-1

A 5 μm pore size ChemoTX plate (Neuroprobe) was used to assess thechemotactic response of THP-1 human monocytic cells. Hybridomasupernatants containing MCP-1 at 10 ng/ml, or RPMI with 10% FBS with orwithout 10 ng/ml chemokine, was added to the lower chamber of the plate.THP-1 cells at 2×10⁶ cells/ml were layered on top. The plate wasincubated for 2 hours at 37° C. in 5% CO₂. The filter was removed andthe number of cells that migrated into the lower chamber was determinedusing Promega Cell Titer reagent. The number of cells was calculatedusing a standard curve (n=4, error bars=standard deviation). The resultsare shown in FIG. 1. FIG. 1 shows that antibodies 11K2, 7F7, 6D21, and7H1 were all able to inhibit MCP-1-induced chemotaxis, although 11K2 and6D21 were the most effective.

II. Characterization of Purified Anti-Chemokine Monoclonal Antibodies

Example 4 Chemokine Specificity and Binding Assays

ELISA specificity assays were performed using purified monoclonalantibodies to confirm the binding specificities of the supernatantMCP-specific monoclonal antibodies described above. Antibodies werepurified by Protein A affinity column chromatography, according tostandard methods known in the art.

ELISA was performed as previously described in Example 1. Briefly,MaxiSorp 384 well plates were coated with 15-20 μl antigen in PBS.Recombinant purified human antigens included: MCP-1, MCP-2, MCP-3,MCP-4, IL-8, eotaxin, murine MCP-1 (JE), murine MCP-3, murine MCP-5, andrat MCP-1. All antigens, including MCP-3, were immobilized. Plates werewashed and purified monoclonal antibody (10 μg/ml) was added to eachwell and incubated for 1 hour at room temperature. Plates were washedand wells were incubated with 20 μl/well of a 1:25,000 dilution of goatanti-mouse IgG peroxidase conjugate (Jackson Catalog Number515-036-003). Plates were incubated for 1 hour at room temperature,washed, and 20 μl/well of substrate (TMB, tetramethylbenzidine, Jackson,Catalog Number 515-036-062) was added. Reaction was allowed to proceedand stopped by addition of 20 μl/well of 2M H₂SO₄. Specificities of thepurified antibodies and are shown in Table 3. Antibodies 1A1 boundspecifically to hMCP-1, hMCP-2, hMCP-3, and mMCP-1. Antibodies 11K2,4N4, 5A13, 6D21, 6I5, and 7H1 bound specifically to hMCP-1, hMCP-2,hMCP-3, mMCP-1, , mMCP-3, and mMCP-5.

TABLE 3 ELISA performed using purified MCP mAbs mMCP-1 hMCP-1 hMCP-2hMCP-3 hMCP-4 (JE) mMCP-3 mMCP-5 HMCP-1 hIL8 hEotaxin 1A1 + + + − + − −− − − 4N4 + + + − + + + − − − 5A13 + + + − + + + − − − 6D21 + + +− + + + − − − 6I5 + + + − + + + − − − 7H1 + + + − + + + − − − 11K2 + + +− + + + − − − D9 + − − − − − − − − − 1M11 + − − − − ND ND ND ND ND3N10 + − − − − ND ND ND ND ND 2O24 + − + − − ND ND ND ND ND 9B11 + − + −− ND ND ND ND ND 9B12 + − + − − ND ND ND ND ND 9C11 + − + − − ND ND NDND ND 5J23 + + +/− − + ND ND − − −

Binding assays were performed using purified monoclonal antibodies toconfirm results obtained with the supernatants. Binding assays wereperformed as described in Example 2. Briefly, ¹²⁵I labeled MCP-1 (2200Ci/mol) was purchased from NEN Life Sciences (Boston, Mass.). Purifiedmonoclonal antibodies at various concentrations (33 nM, 3.3 nM and 0.33nM) were pre-incubated with 1 nM ¹²⁵I MCP-1 (50 μl) for 60 minutes atroom temperature prior to addition of the CCR2-expressing human monocytecell line, THP-1. THP-1 cells (1×10⁷ cells/ml; 50 μl) were resuspendedin binding buffer (50 mM Hepes, 1 mM CaCl₂, 5 mM MgCl₂, 0.5% BSA) andadded to the combination of ¹²⁵I labeled MCP-1 and purified mAb, andincubated at 4° C. for 60 minutes. Cells were then washed 3 times byconfiguration in wash buffer (50 mM Hepes, 1 mM CaCl₂, 5 mM MgCl₂, 500mM NaCl, and 0.5% BSA). Amount of bound ¹²⁵I labeled MCP-1 was thenquantitated for γ-emission. Results from the binding assays are shown inTable 4. This study demonstrates that many of the studied monoclonalantibodies, including 1A1 and 11K2, were effective at blocking hMCP-1binding.

TABLE 4 Purified mAb binding assay Block hMCP-1 cell Antibody binding1A1 + 4N4 + 5A13 + 6D21 + 6I5 + 7H1 + 11K2 + D9 + 1M11 + 3N10 + 2O24 −9B11 − 9B12 − 9C11 − 5J23 +

Example 5 Inhibition of Monocyte Chemotaxis by Anti-Chemokine MonoclonalAntibodies

A. MCP-1 and MCP-2 Chemotaxis Assay

A 5 μm pore size ChemoTX plate (Neuroprobe) was used to assess thechemotactic response of THP-1 human monocytic cells. Purified monoclonalantibodies (100 μg/ml) 11K2, 1A1, D9, and 2O24 were added in combinationwith and without MCP-1 (2.3 nM), MCP-2 (56 nM), and MCP-1/MCP-2 (2.3 nMMCP-1 and 56 nM MCP-2), to the lower chamber of the plate. THP-1 cellsat 2×10⁶ cells/ml were layered on top. The plate was incubated for 4hours at 37° C. in 5% CO₂. The filter was removed and the number ofcells that migrated into the lower chamber was determined using PromegaCell Titer reagent.

The results show that pan-monoclonal antibodies 11K2 and 1A1 wereeffective at inhibiting chemotaxis in the presence of both MCP-1 andMCP-2 (FIG. 2). The results also demonstrate that antibodies D9 and 2O24can inhibit chemotaxis which is induced by MCP-1 alone. Furthermore, asshown in FIGS. 3A and 3B, antibodies 1M11 and 3N10 can inhibit THP-1chemotaxis induced by human MCP-1, and antibody 5J23 can inhibitchemotaxis induced by human and mouse MCP-1. In sum, chemotaxis to thecombination of MCP-1 and MCP-2 is inhibited by 11K2 and 1A1, but is notobserved by antibodies D9 and 2O24, which are MCP-1-specific mAb (D9 and2O24),

Within the pool of monoclonal antibodies studied, there are three groupswhich arise based on their ability to recognize certain MCP antigens.Monoclonal antibodies 1A1 and 11K2 recognize MCP-1, MCP-2 andimmobilized MCP-3. Monoclonal antibodies 1M11 and 3N10 recognize MCP-1,and antibody 2O24 recognizes MCP-1 and MCP-3. Antibody 5J23 recognizesmouse MCP-1 and recognizes only human MCP-1 and human MCP-2.

Results from a separate experiment using the ChemoTX plate (Neuroprobe)assay are shown below in Table 5. The protocol for this experiment wasthe same as previously described, except a titration of mAb was used incombination with fixed MCP concentrations (concentrations of MCPs areshown below in Table 5). The results described in Table 5 demonstratethat mAbs 11K2 and 1A1 are effective at inhibiting huMCP-1, huMCP-2,muMCP-1, and muMCP-5-induced chemotaxis.

TABLE 5 11K2 and 1A1 inhibit THP-1 chemotaxis towards human MCP-1, humanMCP-2, mouse MCP-1 and mouse MCP-5 Human ND₅₀ (nM) MCP-1 MCP-2 MCP-3MCP-4 (2.3 nm) (56 nm) (11.8 nm) (58 nm) Commercial 10.0 33.0  2.6 11.51A1 1.4 47.5 No Inhib No Inhib 11K2 1.3 52.0 No Inhib No Inhib D9 5.8 NoInhib No Inhib No Inhib 2O24 1000.0 No Inhib 143.5 No Inhib Murine ND₅₀(nm) MCP-1 MCP-3 MCP-5 (1.4 nm) MCP-2 (59 nm) MCP-4 (0.54 nm) Commercial3.2 ND 52.0 ND 0.1 1A1 1.7 ND No Inhib ND 13.5 11K2 2.1 ND No Inhib ND19.5 D9 No Inhib ND No Inhib ND No Inhib 2O24 No Inhib ND No Inhib ND NoInhib No inhib = less than 50% Neutralization at 3 umB. Inhibition of Chemotaxis by Cytokines Secreted from RA Fibroblasts

Prior to studying the ability of purified monoclonal antibodies 1A1,11K2, D9, 2O24, and 5D3-F7 (BD Biosciences, Pharmingen, San Diego,Calif.), to inhibit chemotaxis from chemokines secreted from stimulatedRA fibroblasts, a study of the different types of chemokines secreted byRA (rheumatoid arthritis) fibroblasts in response to inflammatorychemokines was performed. RA fibroblasts were exposed for 48 hours to500 U/ml IFN-γ, IFN-γ and 10 ng/ml of IL1β, or media (as a control).Results showed that IFN-γ alone induced low levels of MCP-1, MCP-2,MCP-3, and very low levels of IP10. IFN-γ exposure alone did not induceexpression of Rantes, IL-8, Mip1α, or Mip1β. In contrast, thecombination of IFN-γ and 10 ng/ml of IL1β induced about 27 ng/ml ofMCP-1, 31 ng/ml of MCP-2, 9 ng/ml of MCP-3, and 55 ng/ml of IL-8. Thecombination of IFN-γ and 10 ng/ml of IL1β also yielded low levels ofRantes, IP10, and Mip1α. The media alone control did not induce anychemokine secretion.

The ability of purified monoclonal antibodies to inhibit monocytechemotaxis to cytokines secreted from these stimulated RA fibroblastswas then studied. Supernatant from RA fibroblasts which were exposed toeither media alone, IFN-γ alone, or the combination of IFN-γ and IL-1β,were each tested for their chemotactic ability using human THP-1 cells.As a control, supernatant from unstimulated RA fibroblasts into whichIFN-γ (500 U/ml) and IL1β (10 ng/ml) was spiked was used. Thissupernatant (spite) control was used to evaluate the direct effects ofIL1β and IFN-γ on chemotaxis. As shown in FIG. 4, monocyte chemotaxismediated by cytokines secreted from stimulated RA fibroblasts wasinhibited by MCP mAbs 1A1 and 11K2. MCP-1-specific antibody D9 was alsoeffective at inhibiting chemotaxis in all experimental groups.

Example 6 MCP-1-Induced Calcium Flux Assay for Monoclonal 11K2

The MCP-1-induced calcium flux assay was performed according to standardprocedure. Briefly, monoclonal antibody 11K2 and a chemokine (MCP-1 orMCP-2) were mixed at 200× concentration and pre-incubated for one hour.This mixture was then added to THP-1 cells stirring in a cuvette in afluorimeter at t=30 sec. Calcium flux was measured by a change influorescence of Indo-1. Results show that MCP-1-induced calcium flux inTHP-1 cells was blocked by 11K2 (FIGS. 5A, 5B, and 5C).

Example 7 Agonist Effect of Low Antibody Concentrations of 11K2 and 1A1

A. Chemotaxis Assay

Chemotaxis assays were performed as previously described withrecombinant MCP-2 and using low concentrations of monoclonal antibodies11K2 and 1A1. The results from the chemotaxis assay showed that at a lowconcentration, monoclonal antibodies 11K2 and 1A1 increased MCP-2mediated chemotaxis. As shown in FIG. 6A, there was an increase inchemotaxis observed with low antibody concentrations (ranging from about1-15 nM) of 11K2 and 1A1, in contrast to the MCP-2 mAb 281 (RD Systems,Minneapolis, Minn.). The agonist effect was not seen with the Fabfragment of 11K2 or 1A1, and was MCP-2-specific. As shown in FIG. 6B,low concentrations of the 11K2 Fab fragment did not result in achemotactic increase in response to MCP-2 and showed only antagonistactivity.

B. Calcium Flux Assay

A calcium flux assay was performed as previously described, except a lowconcentration of 11K2 monoclonal antibody (16.5 nM) was also included.The results (FIG. 7B) demonstrate that low concentrations of 11K2exposure results in agonistic activity in a MCP-2 calcium flux assay. Inadditionion, however, 11K2 Fab and F(ab)2 fragments are inhibitory inthe same assay (FIGS. 7A 7C and 7D).

Example 8 Binding Affinity Measurement of Monoclonal 11K2 and 1A1

To measure the affinity of MCP mAb and Fab molecules for soluble MCPmolecules, a kinetic exclusion assay was utilized and affinity measuredusing a KinExA instrument (Sapidyne Instruments Inc., Boise, Id.).

Polymethylmethacrylate beads activated with NHS were coated with 10 μgrecombinant human MCP-1 in 1 ml buffer. The beads were packed into acolumn in the KinExA instrument for each sample. This packed bead columnis able to capture free MCP mAb or Fab flowed through the column. Theamount of free mAb or Fab in solution was determined using a secondarygoat anti-mouse heavy and light chain IgG-Cy5 conjugate.

A fixed amount of 11K2 mAb, 1A1 mAb, 11K2 Fab, or 1A1 Fab was incubatedwith various amounts of human MCP-1, MCP-2 or MCP-3 for three hours. Theamount of uncomplexed free antibody remaining in solution was determinedby flowing these mixtures over the MCP-1-loaded bead column and labelingwith the Cy5 secondary antibody. The fluorescent signal was plottedagainst the MCP concentration and the affinity was determined using aquadratic curve fit

Affinities determined for both 11K2 and 1A1 mAb and Fab molecules arelisted in Table 6 below. Exact affinities of 11K2 mAb and 1A1 mAb forhuman MCP-1 could not be determined, as the affinity is much lower thanthe lowest amount of antibody that can be detected by this method. Inthose cases, only an upper limit to the affinity can be determined.Affinity of the 11K2 Fab and 1A1 Fab for human MCP-3 was not determined(ND).

TABLE 6 MCP binding affinity measurements in solution Antibody MCP-1MCP-2 MCP-3 11K2 mAb  <4 × 10⁻¹³M 1.8 × 10⁻¹¹M >5 × 10⁻⁸M 11K2 Fab 1.1 ×10⁻¹¹M 4.3 × 10⁻¹⁰M ND 1A1 mAb  <7 × 10⁻¹³M 1.2 × 10⁻¹²M >5 × 10⁻⁸M 1A1Fab 1.3 × 10⁻¹¹M 3.2 × 10⁻¹⁰M ND

In sum, monoclonal antibodies 1A1 and 11K2 recognize soluble human MCP-1and MCP-2 with a very high binding affinity which is in the low pMrange. Both 1A1 and 11K2 also recognize mouse MCP-1, while neitherrecognizes soluble MCP-3.

III. Cloning and Sequencing of 1A1 and 11K2 Monoclonal Antibodies

Example 9 Cloning and Sequencing of mu1A1 Variable Regions

Mouse monoclonal antibody 1A1 was cloned and sequenced according to thefollowing procedure. Total cellular RNA from 1A1 murine hybridoma cellswas prepared using the Qiagen RNeasy mini kit according to themanufacturer's recommended protocol.

cDNAs encoding the 1A1 variable regions of the heavy and light chainswere cloned by RT-PCR from total cellular RNA, following standardprocedures known to one of skill in the art. Briefly, following themanufacturer's recommended protocols, first-strand cDNAs (prepared withthe Amersham First-Strand cDNA Synthesis Kit) were amplified by PCRusing the Clontech Advantage 2 PCR Kit. The following primers were usedfor first-strand synthesis of the 1A1 heavy and light chain cDNAs(Y=C/T, and R=A/G): 1A1 Heavy Chain cDNA Primer: 5′-AGG TCT AGA AYC TCCACA CAC AGG RRC CAG TGG ATA GAC-3′ (SEQ ID NO:3) and 1A1 Light ChaincDNA Primer: 5′-GCG TCT AGA ACT GGA TGG TGG GAG ATG GA-3′ (SEQ ID NO:4).

Primers used for PCR amplification of the murine 1A1 immunoglobulinheavy chain variable domain were as follows: 5′-AGG TSM ARC TGC AGS AGTCWG G-3′ (SEQ ID NO:5) and 5′-TGA GGA GAC GGT GAC CGT GGT CCC TTG GCCCC-3′ (SEQ ID NO:6) (S=C/G, M=A/C, R=A/G, and W=A/T). The primers usedfor PCR amplification of the murine 1A1 immunoglobulin light chainvariable domain were: 5′-GAY ATH CAR ATG ACN CAG-3′ (SEQ ID NO:7) and5′-GCG TCT AGA ACT GGA TGG TGG GAG ATG GA-3′ (SEQ ID NO:8) (Y=C/T,H=A/C/T, R=A/G, and N=A/C/G/T).

The PCR was performed at 30 cycles using Clontech's Advantage 2 PCR Kitusing the following PCR conditions: denature 0.5 min at 94° C., anneal 1min at 50° C., and elongate 1 min at 72° C. The PCR products weregel-purified using the Qiagen Qiaquick gel extraction kit following themanufacturer's recommended protocol. Purified 1A1 heavy and light chainPCR products were subcloned into Invitrogen's pCR2.1-TOPO vector usingtheir TOPO TA Cloning kit according to the manufacturer's recommendedprotocol (pCR-049=1A1 heavy chain, pcr-053=1A1 light chain). Insertsfrom multiple independent subclones were sequenced according to methodsknown in the art and those described in Sanger et al., PNAS 74,5463-5467, incorporated herein by reference, and subclones were found tobe identical.

Sequence data was analyzed according to BLAST analysis (seehttp://www.ncbi.nlm.nih.gov). Blast analyses of the variable domainsequences confirmed their immunoglobulin identity. The 1A1 heavy chainvariable domain was determined to be a member of murine subgroup II(C),while the 1A1 light chain variable region was determined to be a memberof murine kappa subgroup II. The predicted amino acid sequences of themature 1A1 murine variable domains, as well as the determined nucleotidesequences, are shown below in Tables 7 and 8.

TABLE 7 Nucleotide sequence of mu1A1 variable domains1A1 Heavy Chain Variable Region (SEQ ID NO: 9) 1GAGGTCCAGCTGCAGCAGTCTGGGGCAGAACTTGTGAGGTCAGGGGCCTCAGTCAAGTTG 60 61TCCTGCACAGCTTCTGGCTTCAACATTAAAGACAACTATATGCACTGGGTGAAGCAGAGG 120 121CCTGAACAGGGCCTGGAGTGGATTGGATGGATTGATCCTGAGAATGGAGATACTGAATAT 180 181GCCCCGAAGTTCCAGGGCAAGGCCACTATGACTGCAGACACATCCTCCAACACAGCCTAC 240 241CTGCAGCTCAGCAGCCTGACATCTGAGGACACTGCCGTCTATTACTGTAATACATGGGCT 300 301TACTACGGTACTAGCTACGGGGGATTTGCTTACTGGGGCCAAGGGACCACGGTCACCGTC 360 361TCCTCA 366 1A1 Light Chain Variable Region (SEQ ID NO: 10) 1GATATCCAGATGACTCAGACTCCACTCACTTTGTCGGTTACCATTGGACAACCAGCCTCC 60 61ATCTCTTGCAAGTCAAGTCAGAGCCTCTTAGATAGTGATGGAAAGACATATTTGAATTGG 120 121TCGTTACAGAGGCCAGGCCAGTCTCCAAAGCGCCTAATCTATCTGGTGTCTAAACTGGAC 180 181TCTGGAGTCCCTGACAGGTTCACTGGCAGTGGATCAGGGACAGATTTCACACTGAAAATC 240 241AGCAGAGTGGAGGCTGAGGATTTGGGAGTTTATTATTGCTGGCAAGGTACACATTTTCCT 300 301CAGACGTTCGGTGGAGGCACCAAGCTGGAGATCAAA 336

TABLE 8 Amino acid sequence of 1A1 variable domains(CDR domains underlined) 1A1 Heavy Chain Variable Region (SEQ ID NO: 11)EVQLQQSGAELVRSGASVKLSCTASGFNIKDNYMHWVKQRPEQGLEWIGWIDPENGDTEYAPK                              CDR1                     CDR2FQGKATMTADTSSNTAYLQLSSLTSEDTAVYYCNTWAYYGTSYGGFAYWGQGTTVTVSS                                      CDR31A1 Light Chain Variable Region (SEQ ID NO: 12)DIQMTQTPLTLSVTIGQPASISCKSSQSLLDSDGKTYLNWSLQRPGQSPKRLIYLVSKLDSGV                           CDR1                        CDR2PDRFTGSGSGTDFTLKISRVEAEDLGVYYCWQGTHFPQTFGGGTKLEIK                                 CDR3

Nucleotide and amino acid comparisons of the 1A1 variable heavy andlight chains are also shown in FIG. 8. The sequence of the CDR regionsof the 1A1 antibody were determined to be the following:

1A1 Heavy Chain Variable Region (SEQ ID NO: 13) CDR1: DNYMH(SEQ ID NO: 14) CDR2: WIDPENGDTEYAPKFQG (SEQ ID NO: 15) CDR3:WAYYGTSYGGFAY 1A1 Light Chain Variable Region (SEQ ID NO: 16) CDR1:KSSQSLLDSDGKTYLN (SEQ ID NO: 17) CDR2: LVSKLDS (SEQ ID NO: 18) CDR3:WQGTHFPQT

Example 10 Cloning and Sequencing of mu11K2 Variable Regions

Mouse monoclonal antibody 11K2 was cloned and sequenced according to thefollowing procedure. Total cellular RNA from 11K2 murine hybridoma cellswas prepared using the Qiagen RNeasy mini kit according to themanufacturer's recommended protocol.

cDNAs encoding the variable regions of the heavy and light chains werecloned by RT-PCR from total cellular RNA. Following the manufacturersrecommended protocols, first-strand cDNAs (prepared with the AmershamFirst-Strand cDNA Synthesis Kit) were amplified by PCR using theClontech Advantage 2 PCR Kit. The following primers were used forfirst-strand synthesis of the 11K2 heavy and light chain cDNAs (Y=C/T,and R=A/G): 11K2 Heavy Chain cDNA Primer 5′- AGG TCT AGA AYC TCC ACA CACAGG RRC CAG TGG ATA GAC-3′ (SEQ ID NO:19) and 11K2 Light Chain cDNAPrimer: 5′-GCG TCT AGA ACT GGA TGG TGG GAG ATG GA-3′ (SEQ ID NO:20).

The primers used for PCR amplification of the murine 11K2 immunoglobulinheavy chain variable domain were: 5′-GGG GAT ATC CAC CAT GGR ATG SAG CTGKGT MAT SCT CTT-3′ (SEQ ID NO:21) and 5′ AGG TCT AGA AYC TCC ACA CAC AGGRRC CAG TGG ATA GAC-3′ (SEQ ID NO:22) (R=A/G, S=C/G, K=G/T, M=A/C, andY=C/T). The primers used for PCR amplification of the murine 11K2immunoglobulin light chain variable domain were: 5′-GAY ATH CAR ATG ACNCAG-3′ (SEQ ID NO:23) and 5′-GCG TCT AGA ACT GGA TGG TGG GAG ATG GA-3′(SEQ ID NO:24) (Y=C/T, H=A/C/T, R=A/G, and N=A/C/G/T).

The PCR was performed at 30 cycles using Clontech's Advantage 2 PCR Kitunder the following PCR conditions: denature 0.5 min at 94° C. anneal 1min at 50° C., and elongate 1 min at 72° C. The PCR products weregel-purified using the Qiagen Qiaquick gel extraction kit following themanufacturer's recommended protocol. Purified 11K2 heavy and light chainPCR products were subcloned into Invitrogen's pCR2.1-TOPO vector usingtheir TOPO TA Cloning kit according to the manufacturer's recommendedprotocol (pCR-008=11K2 heavy chain, pcr-033=11K2 light chain. Insertsfrom multiple independent subclones were sequenced according to methodsknown in the art and those described in Sanger et al., PNAS 74,5463-5467, incorporated herein by reference, and the subclones werefound to be identical.

Sequence data was analyzed according to BLAST analysis (seehttp://www.ncbi.nlm.nih.gov). Blast analyses of the variable domainsequences confirmed their immunoglobulin identity. The 11K2 heavy chainvariable domain was determined to be a member of murine subgroup II(C),while the 11K2 light chain variable region was determined to be a memberof murine kappa subgroup V. The predicted amino acid sequences of themature 11K2 murine variable domains, as well as the determinednucleotide sequences, are shown below in Tables 9 and 10.

TABLE 9 Nucleotide sequence of mu11K2 variable domains11K2 Heavy Chain Variable Region (SEQ ID NO: 25) 1GAGGTTCAGCTGCAGCAGTCTGGGGCAGAGCTTGTGAAGGCAGGGGCCTCAGTCAAGTTG 60 61TCCTGCCCAGCTTCTGGCCTCAACATTAAAGACACCTATATGCACTGGGTGAAGCAGAGG 120 121CCTGAACAGGGCCTGGAGTGGATTGGAAGGATTGATCCTGCGAATGGTAATACTAAATTT 180 181GACCCGAAGTTCCAGGGCAAGGCCACTATAACAGCAGACACATCCTCCAACACAGCCTAC 240 241CTGCAGCTCAGCAGCCTGACATCTGAGGACACTGCCGTCTATTACTGTGCTAGAGGCGTC 300 301TTTGGCTTTTTTGACTACTGGGGCCAAGGCACCACTCTCACAGTCTCCTCA 35111K2 Light Chain Variable Region (SEQ ID NO: 26) 1GACATTCAGATGACTCAGTCTTCATCCTCCTTTTCTGTATCTCTAGGAGACAGAGTCACC 60 61ATTACTTGCAAGGCAACTGAGGACATATATAATCGATTAGCCTGGTATCAGCAGAAACCA 120 121GGAAGTGCTCCTAGGCTCTTAATTTCTGGTGCAACCAGTTTGGAGACTGGGGTTCCTTCA 180 181AGATTCAGTGGCAGTGGATCTGGAAAAGATTACACTCTCAGCATTACCAGTCTTCAGACT 240 241GAGGATGTTGCTACTTATTACTGTCAACAGTTTTGGAGTGCTCCGTACACGTTCGGAGGG 300 301GGGACCAAGCTGGAGATCAAA 321

TABLE 10 Amino acid sequence of 11K2 variable domains(CDR regions underlined) 11K2 heavy chain variable region(SEQ ID NO: 27)EVQLQQSGAELVKAGASVKLSCPASGLNIKDTYMHWVKQRPEQGLEWIGRIDPANGNTKFDPK                              CDR1                     CDR2FQGKATITADTSSNTAYLQLSSLTSEDTAVYYCARGVFGFFDYWGQGTTLTVSS                                     CDR311K2 light chain variable region (SEQ ID NO: 28)DIQMTQSSSSFSVSLGDRVTITCKATEDIYNRLAWYQQKPGSAPRLLISGATSLETGVPSRFS                           CDR1                   CDR2GSGSGKDYTLSITSLQTEDVATYYCQQFWSAPYTFGGGTKLEIK                           CDR3

Nucleotide and amino acid comparisons of the 11K2 variable heavy andlight chains are also shown in FIG. 9. The sequence of the CDR regionsof the 11K2 antibody were determined to be as follows:

11K2 Heavy Chain Variable Region (SEQ ID NO: 29) CDR1: DTYMH(SEQ ID NO: 30) CDR2: RIDPANGNTKFDPKFQG (SEQ ID NO: 31) CDR3: GVFGFFDY11K2 Light Chain Variable Regino (SEQ ID NO: 32) CDR1: KATEDIYNRLA(SEQ ID NO: 33) CDR2: GATSLRT (SEQ ID NO: 34) CDR3: QQFWSAPYT

Based on the results described above, particularly Tables 1 and 3,antibodies were grouped according to their antigen-binding specificity.The CDR region of mAbs which could recognize MCP-1, MCP-2, and MCP-3,including 4N4, 5A13, 6D21, 6I5, 7H1, 11K2, and 1A1 were determined asdescribed above. Sequencing revealed that mAbs 4N4, 5A13, 6D21, 6I5,7H1, and 11K2 all had identical sequences. Monoclonal antibody 1A1 had adifferent sequence. Thus, based on CDR cloning, as well as N-terminalsequencing, two distinct pan-MCP monoclonals antibodies were found toexist.

Forming part of the present disclosure is the appended Sequence Listing,the contents of which are summarized in the table below:

TABLE 11 Sequence listing overview SEQ ID NO: Description Sequence Type1 MCP-1 MRHAS motif amino acid 2 MCP-3 MRHAS motif amino acid 3 1A1Heavy Chain cDNA Primer nucleic acid 4 1A1 Light Chain cDNA Primernucleic acid 5 1A1 Heavy Chain cDNA primer nucleic acid 6 1A1 HeavyChain cDNA primer nucleic acid 7 1A1 Light Chain cDNA primer nucleicacid 8 1A1 Light Chain cDNA primer nucleic acid 9 1A1 Heavy chainvariable region nucleic acid 10 1A1 Light chain variable region nucleicacid 11 1A1 Heavy chain variable region amino acid 12 1A1 Light chainvariable region amino acid 13 1A1 Heavy Chain Variable Region CDR1 aminoacid 14 1A1 Heavy Chain Variable Region CDR2 amino acid 15 1A1 HeavyChain Variable Region CDR3 amino acid 16 1A1 Light Chain Variable RegionCDR1 amino acid 17 1A1 Light Chain Variable Region CDR2 amino acid 181A1 Light Chain Variable Region CDR3 amino acid 19 11K2 Heavy Chain cDNAPrimer nucleic acid 20 11K2 Light Chain cDNA Primer nucleic acid 21 11K2Heavy Chain cDNA Primer nucleic acid 22 11K2 Heavy Chain cDNA Primernucleic acid 23 11K2 Light Chain cDNA Primer nucleic acid 24 11K2 LightChain cDNA Primer nucleic acid 25 11K2 Heavy Chain Variable Regionnucleic acid 26 11K2 Light Chain Variable Region nucleic acid 27 11K2heavy chain variable region amino acid 28 11K2 light chain variableregion amino acid 29 11K2 Heavy Chain Variable Region amino acid CDR1 3011K2 Heavy Chain Variable Region amino acid CDR2 31 11K2 Heavy ChainVariable Region amino acid CDR3 32 11K2 Light Chain Variable Regionamino acid CDR1 33 11K2 Light Chain Variable Region CDR2 amino acid 3411K2 Light Chain Variable Region CDR3 amino acid

One of ordinary skill in the art will recognize that many variations andchanges may be made to the invention as described in the DetailedDescription without departing from the spirit and scope of theinvention. The examples provided herein are merely illustrative, andshould not be construed as limiting of the scope of the invention, whichis set forth in the appended claims. All publications and patentdocuments cited herein, as well as text appearing in the figures andsequence listing, are hereby incorporated by reference in their entiretyfor all purposes to the same extent as if each were so individuallydenoted.

What is claimed is:
 1. A method of reducing chemotaxis in a subjectsuffering from glomerulonephritis, scleroderma, cirrhosis,atherosclerosis, inflammatory bowel disease or rheumatoid arthritis,comprising administering to the subject an isolated antibody or fragmentthereof capable of inhibiting chemotaxis in the subject, wherein saidantibody or fragment thereof binds a plurality of β-chemokines, whereinsaid plurality of β-chemokines comprise MCP-1 and at least one otherβ-chemokine, wherein said antibody or antigen-binding fragmentcomprises: a) a heavy chain variable region having a CDR1 domaincomprising the sequence set forth in SEQ ID NO:29, a CDR2 domaincomprising the sequence set forth in SEQ ID NO:30, and a CDR3 domaincomprising the sequence set forth in SEQ ID NO: 31; and b) a light chainvariable region having a CDR1 domain comprising the sequence set forthin SEQ ID NO:32, a CDR2 domain comprising the sequence set forth in SEQID NO:33, and a CDR3 domain comprising the sequence set forth in SEQ IDNO:34 wherein the isolated antibody is administered in an amountsufficient to reduce the chemotactic activity of at least one of MCP-1,MCP-2, or MCP-3.
 2. The method of claim 1, wherein said antibody orantigen-binding fragment thereof comprises a variable heavy chain regionas set forth in SEQ ID NO:27 and a variable light chain region as setforth in SEQ ID NO:28.
 3. The method of claim 1, wherein the at leastone other β-chemokine is MCP-2.
 4. The method of claim 1, wherein theantibody is a chimeric antibody.
 5. The method of claim 1, wherein theantibody is a humanized antibody.
 6. The method of claim 1, wherein thefragment is an Fab fragment.
 7. The method of claim 1, wherein theantibody is modified by reducing or eliminating at least one potentialglycosylation site.
 8. The method of claim 1, wherein the antibody ismodified by conjugation to a carrier selected from polyethylene glycoland albumen.
 9. The method of claim 1, wherein the constant region ofthe antibody is modified to reduce at least one constant region-mediatedbiological effector function relative to an unmodified antibody selectedfrom the group of binding to an Fc receptor, opsonization, phagocytosis,and antigen-dependent cellular cytotoxicity.
 10. A method of reducingchemotaxis in a subject suffering from glomerulonephritis, scleroderma,cirrhosis, atherosclerosis, inflammatory bowel disease or rheumatoidarthritis, comprising administering to the subject an isolated antibodyor antigen-binding fragment thereof capable of inhibiting chemotaxis inthe subject, wherein said antibody comprises a heavy chain variableregion complementarity determining region (CDR) from an antibody heavychain variable region set forth in SEQ ID NO:27, wherein the isolatedantibody is administered in an amount sufficient to reduce thechemotactic activity of at least one of MCP-1, MCP-2, or MCP-3.
 11. Themethod of claim 10, wherein said antibody or antigen-binding fragmentbinds a plurality of β-chemokines, wherein said plurality ofβ-chemokines comprises MCP-1 and at least one other β-chemokine.
 12. Themethod of claim 11, wherein the at least one other β-chemokine comprisesMCP-2.
 13. A method of reducing chemotaxis in a subject suffering fromglomerulonephritis, scleroderma, cirrhosis, atherosclerosis,inflammatory bowel disease or rheumatoid arthritis, comprisingadministering to the subject an isolated antibody or antigen-bindingfragment thereof capable of inhibiting chemotaxis in the subject,wherein said antibody comprises a light chain variable regioncomplementarity determining region (CDR) from an antibody light chainvariable region as set forth in SEQ ID NO:28, wherein the isolatedantibody is administered in an amount sufficient to reduce thechemotactic activity of at least one of MCP-1, MCP-2, or MCP-3.
 14. Themethod of claim 13, wherein said antibody or antigen-binding fragmentbinds a plurality of β-chemokines, wherein said plurality ofβ-chemokines comprises MCP-1 and at least one other β-chemokine.
 15. Themethod of claim 14, wherein the at least one other β-chemokine comprisesMCP-2.
 16. A method of reducing chemotaxis in a subject suffering fromglomerulonephritis, scleroderma, cirrhosis, atherosclerosis,inflammatory bowel disease or rheumatoid arthritis, comprisingadministering to the subject an isolated single-chain antibodyantigen-binding fragment capable of inhibiting chemotaxis in thesubject, wherein said single-chain antibody fragment specifically bindsMCP-1 and at least one other monocyte chemotactic protein, wherein saidsingle-chain antibody comprises a heavy chain variable regioncomplementarity determining region (CDR) from an antibody heavy chainvariable region set forth in SEQ ID NO:27 or a light chain variableregion complementarity determining region (CDR) from an antibody lightchain variable region set forth in SEQ ID NO:28, wherein saidsingle-chain antibody fragment does not have agonistic chemotacticactivity for MCP-2, wherein the isolated single-chain antibody fragmentis administered in an amount sufficient to reduce the chemotacticactivity of at least one of MCP-1 or MCP-3.
 17. The method of claim 16,wherein the single-chain antibody antigen-binding fragment comprises avariable heavy chain region as set forth in SEQ ID NO: 27 and a variablelight chain region as set forth in SEQ ID NO:28.
 18. The method of claim16, wherein the single-chain antibody antigen-binding fragment binds aplurality of β-chemokines, wherein said plurality of β-chemokinescomprises MCP-1 and at least one other β-chemokine, wherein saidsingle-chain antibody comprises: i. a heavy chain variable region havinga CDR1 domain comprising the sequence set forth in SEQ ID NO:29, a CDR2domain comprising the sequence set forth in SEQ ID NO:30, and a CDR3domain comprising the sequence set forth in SEQ ID NO:31; and ii. alight chain variable region having a CDR1 domain comprising the sequenceset forth in SEQ ID NO:32 a CDR2 domain comprising the sequence setforth in SEQ ID NO:33, and a CDR3 domain comprising the sequence setforth in SEQ ID NO:34.
 19. The method of claim 16, wherein thesingle-chain antibody antigen-binding fragment specifically binds toMCP-1, MCP-2, and MCP-3.
 20. The method of claim 16, wherein thesingle-chain antibody antigen-binding fragment is a monoclonal antibody.21. The method of claim 16, wherein the single-chain antibodyantigen-binding fragment is a humanized antibody.
 22. The method ofclaim 16, wherein the single-chain antibody antigen-binding fragment isselected from the group consisting of an Fab fragment, an Fab' fragment,an F(ab)₂ fragment, and an Fv fragment.
 23. The method of claim 16,wherein the single-chain antibody antigen-binding fragment is an Fabfragment.
 24. The method of claim 16, wherein the single-chain antibodyantigen-binding fragment is conjugated to polyethylene glycol oralbumen.
 25. The method of claim 16, wherein the single-chain antibodyantigen-binding fragment has a Kd for binding affinity to MCP-1 of 1 pMor less or 0.4 pM to about 0.7 pM.
 26. A method of reducing chemotaxisin a subject suffering from glomerulonephritis, scleroderma, cirrhosis,atherosclerosis, inflammatory bowel disease or rheumatoid arthritis,comprising administering to the subject an isolated antibody or fragmentthereof capable of blocking chemotaxis, wherein said antibody orantigen-binding fragment comprises: a) a heavy chain variable regionhaving a CDR1 domain comprising the sequence set forth in SEQ ID NO:29,a CDR2 domain comprising the sequence set forth in SEQ ID NO:30, and aCDR3 domain comprising the sequence set forth in SEQ ID NO: 31; and b) alight chain variable region having a CDR1 domain comprising the sequenceset forth in SEQ ID NO:32, a CDR2 domain comprising the sequence setforth in SEQ ID NO:33, and a CDR3 domain comprising the sequence setforth in SEQ ID NO:34, wherein the isolated antibody is administered inan amount sufficient to reduce the chemotactic activity of at least oneof MCP-1, MCP-2, or MCP-3.